T he cytokine interleukin 1 (IL-1) inhibits contractile responses in rat aorta by causing endothelium-independent and prolonged activation of soluble guanylate cyclase. The present study tested whether IL-1 activates guanylate cyclase by inducing prolonged production of nitric oxide in cultured rat aortic vascular smooth muscle cells (VSMC). IL-1 induced a marked time-dependent increase in cyclic guanosine monophosphate (cGMP) in VSMC which was significant at 6 h, and increased progressively for up to 36 h. This effect of IL-1 was abolished when protein synthesis was inhibited with cycloheximide or actinomycin D, suggesting that the effect of IL-1 involves new protein synthesis. IL-1-induced cGMP accumulation was inhibited by the soluble guanylate cyclase inhibitors, methylene blue, LY83583, and hemoglobin and by the L-arginine analogue NGmonomethyl-L-arginine (L-NMMA). The inhibitory effect of L-NMMA was reversed by a 10-fold excess of L-arginine, but not by D-arginine. Nitrite, an oxidation product of nitric oxide, accumulated in the media of VSMC incubated with IL-1 for 24 h in the presence of L-arginine, whereas both IL-1-induced cGMP accumulation and nitrite production were attenuated in VSMC incubated in L-arginine-deficient medium. In L-arginine-depleted VSMC, IL-1-induced cGMP accumulation was restored to control levels by a 15-min incubation with L-arginine. These results demonstrate that IL-1 activates guanylate cyclase in rat VSMC by inducing production of nitric oxide via a pathway dependent on extracellular L-arginine.