Cryptococcus neoformans is a fungal pathogen that causes meningitis in immunocompromised hosts. The organism has a known sexual cycle, and strains of the MATalpha mating type are more virulent than isogenic MATa strains in mice, and they are more common in the environment and infected hosts. A C. neoformans homolog of the STE12 transcription factor that regulates mating, filamentation, and virulence in Saccharomyces cerevisiae and Candida albicans was identified previously, found to be encoded by a novel region of the MATalpha mating type locus, and shown to enhance filamentous growth when overexpressed. We have disrupted the C. neoformans STE12 gene in a pathogenic serotype A isolate. ste12 mutant strains exhibit a severe defect in filamentation and sporulation (haploid fruiting) in response to nitrogen starvation. In contrast, ste12 mutant strains have only modest mating defects and are fully virulent in two animal models compared to the STE12 wild-type strain. In genetic epistasis experiments, STE12 functions in a MAP kinase cascade to regulate fruiting, but not mating. Thus, the C. neoformans STE12alpha transcription factor homolog plays a specialized function in haploid fruiting, but it is dispensable or redundant for mating and virulence. The association of the MATalpha locus with virulence may involve additional genes, and other transcription factors that regulate mating and virulence remain to be identified.