The modulating action and mechanism of endogenous nitric oxide (NO) on the delayed rectifier potassium currents in cultured hippocampal neurons were examined using whole-cell patch clamp techniques. L-arginine (L-Arg, 2 mmol/L), a substrate of NO synthases, significantly suppressed the delayed rectifier K+ currents in hippocampal neurons, while its isomer D-arginine (D-Arg, 2 mmol/L) exerted no effect. Moreover, pretreatment with NO synthase inhibitor L-NAME (0.5 mmol/L) completely blocked the suppressing effect by L-Arg, indicating that L-Arg exerted its modulation by producing NO but not by itself. No effect was found on the L-Arg-induced inhibition by 10 min pretreatment of 10 μmol/L ODQ (a specific inhibitor of guanylate cyclase). In contrast, thiol-alkylating agent N-ethylmaleimide (1 mmol/L) completely precluded L-Arg-induced inhibition on the whole K+ currents. The results indicate that endogenous NO modulates the delayed rectifier K+ currents in cultured hippocampal neurons mostly through S-nitrosylation.