In the budding yeast Saccharomyces cerevisiae, the process of conjugation of haploid cells of genotype MATa and MAT alpha to form MATa/alpha diploids is triggered by pheromones produced by each mating type. These pheromones stimulate a cellular response by interaction with receptors linked to a heterotrimeric G protein. Although genetic analysis indicates that the pheromone signal is transmitted through the G beta gamma dimer, the initial target(s) of G protein activation remain to be determined. Temperature-sensitive cells with mutations of the CDC24 and CDC42 genes, which are incapable of budding and of generating cell polarity at the restrictive temperature, are also unable to mate. Cdc24 acts as a guanylyl-nucleotide-exchange factor for the Rho-type GTPase Cdc42, which has been shown to be a fundamental component of the molecular machinery controlling morphogenesis in eukaryotic cells. Therefore, the inability of cdc24 and cdc42 mutants to mate has been presumed to be due to a requirement for generation of cell polarity and related morphogenetic events during conjugation. But here we show that Cdc42 has a direct signalling role in the mating-pheromone response between the G protein and the downstream protein kinase cascade.