The STE3 gene of Saccharomyces cerevisiae encodes a G protein-coupled receptor that is specific for the mating pheromone a-factor. The ste3L194Q mutation, which leads to the substitution of glutamine for leucine-194 within the third cytoplasmic loop of the receptor, resulted in a 20-fold increase in pheromone sensitivity and also caused partial constitutive activation of the response pathway. Moreover, other amino acid substitutions at the 194 position and several deletion mutations that collectively remove most of the third cytoplasmic loop resulted in hyperactive receptors. Therefore, we suggest that one role of the third cytoplasmic loop is to function as a negative regulatory domain involved in the maintenance of a nonsignaling state of the receptor. The constitutive activity and the pheromone hypersensitivity of ste3L194Q cells were recessive, suggesting that the wild-type receptor can antagonize the signal associated with the activated receptor. The ste3 delta 306 mutation, which results in truncation of most of the C-terminal domain of the receptor, led to a 20-fold increase in pheromone sensitivity, indicating that this domain also mediates negative regulation of the receptor. The ste3L194Q and ste3 delta 306 mutations appear to affect receptor activity independently, because the double mutant was associated with a 400-fold increase in pheromone sensitivity.