To study the effects of chronic in vivo inhibition of NO synthase on endothelium-dependent hyperpolarization, cell-membrane potential (in individual vascular smooth-muscle cells) and changes in tension (in isolated rings) were recorded from isolated canine coronary arteries and guinea-pig carotid arteries and aortas. In coronary arteries taken from control dogs and contracted with U46619, acetylcholine- and bradykinin-induced endothelium-dependent relaxations, which were unaffected by short-term in vitro exposure to indomethacin but were inhibited partially by L-nitro-arginine (LNA). In coronary arteries taken from dogs treated over the long term in vivo with LNA (30 mg/kg on the first day and 20 mg/kg the 7 following days, i.v.), the response to acetylcholine and bradykinin was inhibited when compared with arteries from control dogs. Short-term in vitro exposure to LNA or indomethacin or both did not influence the effects of either agonist. In these arteries, the hyperpolarizing response to acetylcholine, observed in the presence of LNA and indomethacin, was enhanced, whereas that to bradykinin was partially inhibited. In the guinea pig isolated aorta, the relaxation to bradykinin was abolished by long-term in vivo treatment with L-nitro-arginine-methyl-ester (L-NAME; 1.5 mg/ml, in the drinking water for > or =4 days). In the isolated guinea pig carotid artery studied in the presence of LNA and indomethacin, acetylcholine induced a hyperpolarization that was not significantly affected by long-term in vivo treatment with L-NAME. These findings indicate that endothelium-dependent hyperpolarizations are maintained during long-term inhibition of NO synthase and probably act as a back-up mechanism to elicit endothelium-dependent relaxations.