The mating response pathway of the yeast Saccharomyces cerevisiae includes a heterotrimeric guanine nucleotide-binding protein (G protein) that activates a mitogen-activated protein (MAP) kinase cascade by an unknown mechanism. An amino-terminal fragment of the MAP kinase scaffold protein Ste5p that interfered with pheromone-induced cell cycle arrest was identified. A haploid-specific interaction between the amino terminus of Ste5p and the G protein beta subunit Ste4p was also detected in a two-hybrid assay, and the product of a signaling-defective allele of STE4 was defective in this interaction. In cells with a constitutively activated pheromone response pathway, epitope-tagged Ste4p was coimmunoprecipitated with Ste5p, Thus, association of the G protein and the MAP kinase cassette via the scaffolding protein Ste5p may transmit the G protein signal.