Sexual reproduction is nearly universal and yet occurs in myriad forms throughout nature. We are addressing sex in the fungal kingdom (1) focusing on three general themes. First, the structure, function, and evolution of the mating type locus (MAT) that governs cell identity during sexual reproduction, and which serves as a model for the evolution of gene clusters and mirrors steps in sex chromosome evolution. Second, how transitions in sexual reproduction occur between out-crossing tetrapolar and inbreeding bipolar mating systems, and heterothallic and homothallic mating, including the emergence of unisexual reproduction in some species. Third, we are exploring the evolutionary trajectory of Candida pathogenic species in which some have retained complete sexual cycles, including meiosis, whereas others have adopted parasexual cycles in which meiosis is absent or cryptic. In the Cryptococcus neoformans pathogenic species complex, an ancestral tetrapolar mating type locus is hypothesized to have expanded via gene acquisition into two unlinked sex determinants which then fused, forming an unusually large >100 kb bipolar MAT locus linked to differentiation and virulence. Key aspects of this model are being tested in closely related species, (C. amylolentus, C. heveanensis). This analysis reveals extant species that harbor two large unlinked MAT gene clusters corresponding to the homeodomain and pheromone/receptor gene clusters of the Cryptococcus MAT locus prior to fusion to a linked, bipolar sex determinant. To extend our understanding of MAT evolution, the sex locus has been defined in representative zygomycete basal fungi (Phycomyces, Mucor, Rhizopus, E. cuniculi). This reveals a small, conserved sex locus encoding divergent HMG domain proteins that dictate sexual identity, withimplications for the evolutionary trajectory of MAT throughout the fungal kingdom and the evolution of HMG based sex determination as an ancestral form. Transitions in modes of sexual reproduction commonly occur throughout the fungal kingdom between heterothallic and homothallic species, and bipolar and tetrapolar sex determination. Our studies reveal a novel form of homothallic unisexual reproduction in C. neoformans involving only one of the two mating types, and address where and how this impacts a largely unisexual population structure. An extant sexual cycle has been discovered for the sibling species C. amylolentus. Sex occurs between strains of opposite mating type yet generates predominantly sterile isolates mixed with a minority of fertile isolates largely of only one mating type. Thus, this modified sexual cycle appears to benefit predominantly only one of the two mating types in the population, and may be the antecedent in transition to unisexual mating. Finally, genetically engineering Cryptococcus strains to represent the hypothesized tetrapolar ancestor and tripolar intermediate provides insights into molecular mechanisms by which transitions can occur between bipolar and tetrapolar sex determination, and may provide insights into how tetrapolar sex first evolved in the ancestral basidiomycete lineage, and the plasticity of sex determination. The most common human fungal pathogen Candida albicans is an obligate diploid that was thought to be asexual until the recent discovery of the mating type locus and an extant parasexual cycle. In contrast, other pathogenic Candida species (C. lusitaniae, C. guilliermondii) are haploid with complete sexual cycles, including sporulation. Our studies define the structure and function of MAT in these species, and genomic analysis reveals that many key meiotic genes were lost. We find that meiosis still occurs during mating in C. lusitaniae, yet generates a high level of aneuploidy, possibly as an additional route to genetic diversity. Features of this modified sexual cycle may provide insights into the sexual nature of C. albicans. (1) Sex in Fungi: Molecular Determination and Evolutionary Implications, ASM press, 2007.