The opportunistic fungal pathogen Cryptococcus neoformans has two mating types, MATa and MAT alpha. The MAT alpha strains are more virulent. Mating of opposite mating type haploid yeast cells results in the production of a filamentous hyphal phase. The MAT alpha locus has been isolated in this study in order to identify the genetic differences between mating types and their contribution to virulence. A 138-bp fragment of MAT alpha-specific DNA which cosegregates with alpha-mating type was isolated by using a difference cloning method. Overlapping phage and cosmid clones spanning the entire MAT alpha locus were isolated by using this MAT alpha-specific fragment as a probe. Mapping of these clones physically defined the MAT alpha locus to a 35- to 45-kb region which is present only in MAT alpha strains. Transformation studies with fragments of the MAT alpha locus identified a 2.1-kb XbaI-HindIII fragment that directs starvation-induced filament formation in MATa cells but not in MAT alpha cells. This 2.1-kb fragment contains a gene, MF alpha, with a small open reading frame encoding a pheromone precursor similar to the lipoprotein mating factors found in Saccharomyces cerevisiae, Ustilago maydis, and Schizosaccharomyces pombe. The ability of the MATa cells to express, process, and secrete the MAT alpha pheromone in response to starvation suggests similar mechanisms for these processes in both cell types. These results also suggest that the production of pheromone is under a type of nutritional control shared by the two cell types.