The STE5 gene encodes an essential element of the pheromone response pathway which is known to act either after the G subunit encoded by the STE4 gene or at the same step. Mutations in STE5, designated STE5Hyp, that partially activate the pathway in the absence of pheromone were isolated. One allele (STE5Hyp-2) was shown to cause a single amino acid substitution near the N terminus of the predicted STE5 protein. Immunoblotting with anti-Ste5 antibodies indicated that the phenotype was not due to an increased level of the mutant STE5 protein. A multicopy episomal plasmid containing a STE5Hyp allele partially suppressed both the block in pheromone-inducible transcription and the sterility phenotype caused by null alleles of the STE2, STE4, or STE18 gene, indicating that the STE5 product acts after the receptor (STE2 product) and after the G protein beta and gamma subunits (STE4 and STE18 products, respectively). However, the phenotypes of the STE5Hyp mutations were less pronounced in ste4 and ste18 mutants, suggesting that the STE5Hyp-generated signal partially depends on the proposed G beta gamma complex. The STE5Hyp alleles did not suppress ste7, ste11, ste12, or fus3 kss1 null mutants, consistent with previous findings that the STE5 product acts before the protein kinases encoded by STE7, STE11, FUS3, and KSS1 and the transcription factor encoded by STE12. The mating defects of the ste2 deletion mutant and the temperature-sensitive ste4-3 mutant were also suppressed by overexpression of wild-type STE5. The slow-growth phenotype manifested by cells carrying STE5Hyp alleles was enhanced by the sst2-1 mutation; this effect was eliminated in ste4 mutants. These results provide the first evidence that the STE5 gene product performs its function after the G protein subunits.