The exact mechanisms controlling nitric oxide synthase (NOS) activity within enteric neurons are largely unknown. In this study, the effect of exogenous nitric oxide (NO) on NOS activity was investigated in enteric synaptosomes of rat ileum. 3-Morpholinosydnonimine (SIN-1; 10⁴ M) and S-nitroso-N-acetylpenicillamine (SNAP; 10⁴ M) significantly inhibited NOS activity by 53% and 48%, respectively. However, superoxide dismutase (SOD; 160 U/ml) as well as the NO scavenger oxyhemoglobin (10³ M) did not influence NO donor-induced inhibition. In contrast, the inhibitory effect was antagonized by diethyldithiocarbamate (3 × 10⁴ M), an inhibitor of endogenous Cu/Zn SOD. Inhibition of NOS by exogenous NO was dependent on glutathione (GSH), since the inhibitory effect was augmented in the presence of GSH (5 × 10⁴ M) and antagonized by the GSH-depletor DL-buthionine-SR-sulfoximine (5 × 10⁴ M), suggesting that NO might be protected from extracellular breakdown by reaction with GSH. The reaction product of SIN-1/SNAP and GSH was identified as a nitrosothiol. In the presence of the Cu⁺-chelator neocuproine (10⁵ M), inhibition of NOS by SNAP/SIN-1 was reversed, suggesting that nitrosothiol formation is intermediary. These findings are indicative of a feedback inhibition of enteric NOS, presumably via formation of a nitrosothiol intermediate.