In mating mixtures of Saccharomyces cerevisiae, cells polarize their growth toward their conjugation partners along a pheromone gradient. This chemotropic phenomenon is mediated by structural proteins such as Far1 and Bem1 and by signaling proteins such as Cdc24, Cdc42, and Gbetagamma. The Gbetagamma subunit is thought to provide a positional cue that recruits the polarity establishment proteins, and thereby induces polarization of the actin cytoskeleton. We identified RHO1 in a screen for allele-specific high-copy suppressors of Gbetagamma overexpression, suggesting that Rho1 binds Gbetagamma in vivo. Inactivation of Rho1 GTPase activity augmented the rescue phenotype, suggesting that it is the activated form of Rho1 that binds Gbetagamma. We also found, in a pull-down assay, that Rho1 associates with GST-Ste4 and that Rho1 is localized to the neck and tip of mating projections. Moreover, a mutation in STE4 that disrupts Gbetagamma-Rho1 interaction reduces the projection tip localization of Rho1 and compromises the integrity of pheromone-treated cells deficient in Rho1 activity. In addition to its roles as a positive regulator of 1,3-beta-glucan synthase and of the cell integrity MAP kinase cascade, it was recently shown that Rho1 is necessary for the formation of mating projections. Together, these results suggest that Gbetagamma recruits Rho1 to the site of polarized growth during mating.