Abstract: In this work, we have studied the effects of pure nitric oxide (NO) on the regulation of catecholamine (CA) secretion by chromaffin cells, as well as the possible presence of its synthesizing enzyme l-arginine:NO synthase (NOS) in these cells. Our results show that NO produces a large stimulation of basal CA secretion. This effect was calcium- and concentration-dependent (EC50 = 64 ± 8 µM) and was not due to nonspecific damage of the tissue by NO. NO also modulates the CA secretion evoked by nicotine in a dose-dependent manner. Although it has a stimulatory effect on the CA secretion evoked by low doses of nicotine (<3 µM; EC50 = 16 ± 3 µM), it produces a dose-dependent inhibition of the CA secretion induced by high doses of nicotine (≥30 µM; IC50 = 52 ± 6 µM). The mechanism by which NO modulates CA secretion seems to be through the increase in the cyclic GMP levels, because there was a close correlation between the CA secretion and the cyclic GMP levels. The presence of a specific activity of NOS in chromaffin cells has been demonstrated by two independent methods: release of [14C]citruiline from [14C]arginine and formation of an NO-hemoglobin complex. NOS activity was about 0.5 pmol/min/mg of protein. It was calcium- and mainly calmodulin-dependent and could be specifically blocked by the NOS inhibitor N-methyl-l-arginine. These results suggest that NO could be an important intracellular messenger in the regulation of neurosecretion in chromaffin cells.