Cryptococcus neoformans is an important human pathogenic fungus with a defined sexual cycle and well-developed molecular and genetic approaches. C. neoformans is predominantly haploid and has two mating types, MATa and MATα. Mating is known to be regulated by nutritional limitation and thought also to be regulated by pheromones. Previously, a portion of the MATα locus was cloned, and a presumptive pheromone gene, MFα1, was identified by its ability to induce conjugation tube-like filaments when introduced by transformation into MATa cells. Here, the ability of the MFα1 gene to induce these morphological changes in MATa cells was used as a phenotypic assay to perform a structure-function analysis of the gene. We show that the MFα1 open reading frame is required for the morphological response of MATa cells. We also find that the cysteine residue of the C-terminal CAAX motif is required for activity of the MFα1 pheromone. In addition, we use a reporter system to measure the expression levels of the MFα1 pheromone gene and find that two signals, nutrient starvation and the presence of factors secreted by mating partner cells, impinge on this promoter and regulate MFα1 expression. We identify a second pheromone gene, MFα2, and show phenotypically that this gene is also expressed. Finally, we have synthesized the MFα1 pheromone and show that only the predicted mature modified form of the α-factor peptide triggers morphological responses in MATa cells.