The objective of this study was to determine whether nitric oxide (NO) could function as a negative feedback modulator of endothelial cell function by inhibiting NO synthase in vascular endothelial cells. The rationale for this approach was a previous study from this laboratory, which revealed that NO inhibits neuronal NO synthase from rat cerebellum. In the present study, NO and NO-donor agents noncompetitively inhibited NO synthase derived from bovine aortic endothelial cells. Oxyhemoglobin blocked the inhibitory action of NO and by itself increased NO synthase activity. This finding suggests that NO acts as a negative feedback modulator of NO synthase. In intact aortic endothelial cells grown on microcarrier beads and perfused in a bioassay cascade system, pretreatment of cells with NO-donor agents caused a marked inhibition of endothelial NO biosynthesis in response to bradykinin and increased fluid shear or flow. When isolated bovine pulmonary arterial rings precontracted by phenylephrine were used, pretreatment of arterial rings with NO-donor agents diminished endothelium-dependent arterial relaxation involving the L-arginine-NO pathway without altering endothelium-independent relaxation to NO itself. On the basis of these studies, NO is suggested to play an important negative feedback regulatory role on endothelial NO synthase and, therefore, vascular endothelial cell function.