Muscle strips loaded with the Ca2+ indicator aequorin were studied in vitro to determine the effects of inhibitory stimuli on force and cytosolic free Ca2+. In muscles contracted isometrically with acetylcholine (ACh), 5-hydroxytryptamine (5-HT), carbachol, decreases in muscle force caused by isoproterenol (10(-5) M) or forskolin (10(-5) M) were accompanied by proportional decreases in aequorin luminescence. A similar relationship between decreases in muscle force and aequorin luminescence was observed when muscles were relaxed by stimulating Na+-K+-ATPase activity. These results suggest that the Ca2+ sensitivity of contractile proteins was not decreased during adenosine 3',5'-cyclic monophosphate (cAMP)-dependent relaxation. However, aequorin luminescence did not decrease when muscles contracted by K+ depolarization were relaxed with isoproterenol. Incubation of muscles in forskolin depressed increases in both force and aequorin luminescence in response to 5-HT or ACh. Incubation of muscles in isoproterenol had a similar effect on responses to 5-HT but depressed increases in force without depressing increases in luminescence in response to ACh. Results indicate that under most conditions the reduction of cytosolic Ca2+ plays an important role in the cAMP-dependent relaxation of canine tracheal smooth muscle.