Relaxation of the trabecular smooth muscle of the corpus cavernosum (the erectile tissue) of the penis is mediated by nitric oxide released by the nerves and endothelium. We have investigated the physiological role of oxygen tension in the regulation of trabecular smooth muscle tone. In human subjects, measurement of intracavernosal PO2 in blood drawn from corpus cavernosum in the flaccid state was comparable to that of venous blood (25-43 mmHg). Vasodilatation of the resistance arteries and trabecular smooth muscle relaxation by intracavernosal injection of papaverine and phentolamine caused oxygen tension to rise rapidly to arterial levels (PO2 approximately 100 mmHg). Isolated human and rabbit corpus cavernosum tissue strips in organ baths, exposed to arterial-like PO2 relaxed to the endothelium-dependent dilator acetylcholine and to electrical stimulation of the autonomic dilator nerves. These nitric oxide-mediated responses were progressively inhibited as a function of decreasing PO2 to levels measured in the flaccid penis. Reverting to normoxic conditions readily restored endothelium-dependent and neurogenic relaxation. Relaxation to exogenous nitric oxide was not impaired in low PO2. In rabbit corpus cavernosum, low PO2 reduced basal levels of cGMP and prevented cGMP accumulation induced by stimulation of dilator nerves. Furthermore, low PO2 inhibited nitric oxide synthase activity in corpus cavernosum cytosol. It is concluded that physiological concentrations of oxygen modulate penile erection by regulating nitric oxide synthesis in corpus cavernosum tissue.