Smooth muscle cells were isolated from the dog gallbladder and used to examine whether bombesin-14 and substance P cause direct contraction and whether this effect is mediated by the same or different receptors. Both peptides caused concentration-dependent contraction that was inhibited by two substance P derivatives, [D-Pro2,D-Trp7,9]SP ([PTT]SP) and [D-Arg1,D-Pro2,D-Trp7,9,Leu11]SP ([APTTL]SP). In nonmuscle cells, these substance P derivatives exhibit preferential antagonism of responses to substance P and bombesin, respectively; accordingly, these properties were used to demonstrate the presence of separate bombesin and substance P receptors. Estimates of the apparent Ki showed that [PTT]SP was 40 times more potent than [APTTL]SP as an antagonist of the response to substance P, whereas [APTTL]SP was 85 times more potent than [PTT]SP as an antagonist of the response to bombesin-14. Estimates of median inhibitory concentration obtained from inhibition curves for the effect of various concentrations of each antagonist on the responses to substance P and bombesin-14 confirmed that [PTT]SP was a preferential antagonist of the response to substance P and [APTTL]SP, a preferential antagonist of the response to bombesin-14. The results demonstrate the presence of separate bombesin and substance P receptors mediating contraction of smooth muscle cells of the dog gallbladder.