1. Membrane potential was recorded with intracellular microelectrodes from the smooth muscle of coronary arteries of guinea-pigs, and the responses to endothelium-derived relaxants were studied under a variety of conditions. 2. Stimulation of the endothelium with brief applications of acetylcholine or substance P evoked concentration-dependent hyperpolarizations that were complex in nature. A transient component, which is likely to result from endothelium-derived hyperpolarizing factor (EDHF), was followed by a slow component that resulted from the production of nitric oxide (NO) and a prostaglandin. 3. The ability of exogenous and endogenous NO and prostacyclin to hyperpolarize the membrane depended upon the smooth muscle being under stretch. Unstretched preparations responded to acetylcholine with only the transient component of hyperpolarization; NO and prostacyclin were without effect. 4. In stretched preparations exogenous NO and prostacyclin, and its synthetic analogue methyl prostacyclin (Iloprost), evoked hyperpolarization, and the slow component of the response induced by acetylcholine appeared. The amplitudes of these responses reached maximum when the tissues were stretched to the equivalent of approximately 50 mmHg. 5. From a resting membrane potential of -61 +/- 0.6 mV, exogenous NO and Iloprost hyperpolarized the smooth muscle to around -80 mV. The EC50 values for NO- and Iloprost-induced hyperpolarization were 2.6 x 10(-6) and 1.3 x 10(-8) M, respectively. 6. Coronary arterial smooth muscles from rats, rabbits and sheep also hyperpolarized in response to exogenous NO, although their sensitivities were less than those of preparations obtained from guinea-pigs. Iloprost hyperpolarized tissues from rabbits and sheep but not those obtained from rats. 7. It is concluded that the endothelial lining of coronary arteries can release three factors, EDHF, NO and prostacyclin, all of which can hyperpolarize the membrane of the smooth muscle. The relative proportions and significance of each factor depends on the amount of stretch, on the artery and on the species of animal.