The purpose of this study was to investigate the effects of epinephrine on neurotransmission mediated by cholinergic neurons derived from the rat retina. We used a culture system in which striated muscle cells served as postsynaptic targets for cholinergic neurons of the embryonic retina. This culture system permitted the physiological monitoring of acetylcholine released by retinal neurons. Here, we report that epinephrine facilitates evoked transmission across retina-muscle synapses. This facilitation of cholinergic transmission by epinephrine is reversible, can be mimicked by isoproterenol (a beta adrenoceptor agonist) and blocked by propranolol (a beta adrenoceptor antagonist). Neither the alpha-2 adrenoceptor blocker, yohimbine, nor the dopamine receptor antagonist, haloperidol, blocked this effect of epinephrine. Since epinephrine was found not to influence the membrane potential of muscle cells nor the responses of myotubes to acetylcholine, epinephrine appeared to have mediated its facilitatory effect on cholinergic transmission by affecting retinal cells. Because previous findings indicated that adenosine 3',5'-cyclic monophosphate may be involved in the modulation of transmission at retina-muscle synapses, the effect of epinephrine on adenosine 3',5'-cyclic monophosphate levels was investigated. Our biochemical studies demonstrated that epinephrine could increase adenosine 3',5'-cyclic monophosphate levels markedly in cultured retinal cells. The accumulation of adenosine 3',5'-cyclic monophosphate induced by epinephrine could be blocked by propranolol, but not by yohimbine nor haloperidol. Taken together, the results indicate that the facilitatory effect of epinephrine is mediated via a beta adrenoceptor and may involve an increase in adenosine 3',5'-cyclic monophosphate levels. Our findings are in agreement with the hypothesis that epinephrine may be a modulatory neurotransmitter in the rat retina.