Isometric tension changes of the bovine iris dilator muscle were investigated in vitro. Electrical field stimulation (0.03-1 msec duration) produced a relaxation, which was abolished by the addition of tetrodotoxin, thereby suggesting a neuronal origin. Marked relaxation was also initiated in high K solution. Both of these relaxations were potentiated by neostigmine, while atropine abolished the relaxations. Exogenous application of acetylcholine or carbachol produced dose-dependent relaxations that were not antagonized by adrenoceptor blocking agents. These observations indicate that the relaxations induced either by electrical nerve stimulation or by high K are mainly mediated via excitation of cholinergic nerves and that the consequent release of acetylcholine relaxes the muscle. The cholinergic inhibitory mechanism may be accompanied by hyperpolarization of the muscle membrane or be due to a direct transmitter action via "pharmacomechanical coupling." Adrenergic agonists and high K produced weak contractions even when the muscle relaxed with carbachol application. The adrenergic system and the depolarization of the muscle seem to play some role on the motor function of the bovine dilator. The present experiment suggests that the cholinergic system plays an unexpectedly dominant part in dilation of the bovine pupil, since cholinergic agents produced considerable responses of the dilator muscle, as compared to findings in the case of adrenergic agonists. The inhibitory cholinergic nerves innervating the bovine dilator are closely related to the miotic action of the sphincter muscle. The unexpected inhibitory action of acetylcholine may possibly be a general occurrence among mammals.