The neuronal and endothelial isoforms of nitric oxide (NO) synthase (nNOS and eNOS, respectively) both catalyze the production of NO but are regulated differently. Stably transfected HEK 293 cell lines containing nNOS, eNOS, and a soluble mutant of eNOS were therefore established to compare their activity in a common cellular environment. NOS activity was determined by measuring L-[3H]citrulline production in homogenates and intact cells, the conversion of oxyhemoglobin to methemoglobin, and the production of cGMP. The results indicate that nNOS is more active than eNOS, both in unstimulated as well as calcium-stimulated cells. Under basal conditions, the soluble mutant of eNOS appeared to be slightly more active than wild-type eNOS in terms of NO and cGMP formation, suggesting that membrane association may be crucial for inhibition of basal NO release but is not required for stimulation by Ca2+-mobilizing agents. The maximal activity of soluble guanylate cyclase was significantly reduced by transfection with wild-type eNOS due to downregulation of mRNA expression. These results demonstrate that nNOS and eNOS behave differently even in an identical cellular environment.