Partial sequence analysis of the Cryptococcus neoformans MATalpha mating type locus revealed the presence of a gene with substantial sequence similarity to other fungal mitogen-activated protein (MAP) kinase kinase kinase (MAPKKK) genes. The C. neoformans gene, designated STE11alpha, showed the highest degree of similarity to the Neurospora crassa nrc-1, Schizosaccharomyces pombe byr2 and Saccharomyces cerevisiae STE11 genes. A polymerase chain reaction-mediated sib-selection technique was successfully adapted for the purpose of disrupting STE11alpha. C. neoformans ste11alphaDelta mutants were found to be sterile, consistent with the phenotypes of ste11 and byr2 mutants in S. cerevisiae and S. pombe respectively. Haploid ste11alphaDelta mutants were also found to be unable to produce hyphae, suggesting that the C. neoformans gene is functionally conserved when compared with its S. cerevisiae MAPKKK counterpart. Comparison of the wild-type STE11alpha strain with a ste11alphaDelta disruptant for virulence using the mouse model showed that the ste11alphaDelta strain was less virulent, but the difference was only minor. In spite of some of the conserved functions of STE11alpha, linkage analysis showed that STE11alpha is only found in mating type alpha strains. These results demonstrate that, although functionally conserved, the mating pathway in C. neoformans has a unique organization.