The effect of Na-K adenosinetriphosphatase (ATPase) on relaxation induced by isoproterenol, prostaglandin E2, sodium nitroprusside, and forskolin, a specific stimulant of adenylate cyclase, was investigated in canine tracheal smooth muscle strips. Relaxation in response to isoproterenol, prostaglandin E2, and forskolin was significantly decreased after inhibition of the Na-K ATPase by ouabain or a potassium-free medium, but relaxation to sodium nitroprusside was not affected. Relaxation to isoproterenol was greater in muscles contracted by 5-hydroxytryptamine than in those contracted by acetylcholine. The stimulation of Na-K ATPase activity with potassium also caused differences in relaxation between tissues contracted with 5-hydroxytryptamine or acetylcholine. Relaxation caused by isoproterenol by activation of the Na-K-ATPase was also decreased by the Ca2+-channel antagonists, verapamil and diltiazem. The results suggest 1) Na-K ATPase activity modulates relaxation caused by isoproterenol, prostaglandin E2, and forskolin in canine tracheal smooth muscle, 2) isoproterenol or activation of the Na-K ATPase may cause relaxation partly by reducing Ca2+ influx through potential-dependent Ca2+ channels, and 3) the differences in the inhibitory effects of isoproterenol and Na-K ATPase activity on muscles contracted by acetylcholine and 5-hydroxytryptamine could be due to differences between these contractile agents in their dependence on extracellular Ca2+ for activation.