A 14-mer α-pheromone peptide of Candida albicans was chemically synthesized and used to analyze the role of white-opaque switching in the mating process. The α-pheromone peptide blocked cell multiplication and induced “shmooing” in a/a cells expressing the opaque-phase phenotype but not in a/a cells expressing the white-phase phenotype. The α-pheromone peptide induced these effects at 25°C but not at 37°C. An analysis of mating-associated gene expression revealed several categories of gene regulation, including (i) MTL-homozygous-specific, pheromone stimulated, switching-independent (CAG1 and STE4); (ii) mating type-specific, pheromone-induced, switching-independent (STE2); and (iii) pheromone-induced, switching-dependent (FIG1, KAR4, and HWP1). An analysis of switching-regulated genes revealed an additional category of opaque-phase-specific genes that are downregulated by α-pheromone only in a/a cells (OP4, SAP1, and SAP3). These results demonstrate that α-pheromone causes shmooing, the initial step in the mating process, only in a/a cells expressing the opaque phenotype and only at temperatures below that in the human host. These results further demonstrate that although some mating-associated genes are stimulated by the α-pheromone peptide in both white- and opaque-phase cells, others are stimulated only in opaque-phase cells, revealing a category of gene regulation unique to C. albicans in which α-pheromone induction requires the white-opaque transition. These results demonstrate that in C. albicans, the mating process and associated gene regulation must be examined within the context of white-opaque switching.