Endothelial cell (EC)–released agents are active regulators of vascular smooth muscle cell (VSMC) functions. The first aim of the present work was to analyze the effect of ECs on interleukin-1β (IL-1β)–induced NO production by SMCs. Bovine aortic ECs (BAECs) and BVSMCs in culture were used for the study. IL-1β (0.03 U/L) stimulated nitrite production by BVSMCs. This increase was smaller in the presence of BAECs. This effect was accompanied by reduced expression of inducible NO synthase (iNOS) in BVSMCs coincubated with BAECs, as analyzed by Western blot analysis. The reduction in iNOS protein expression was partially reversed by a polyclonal antibody against transforming growth factor-β (TGF-β). Furthermore, we examined the cytotoxic effect of the NO released from BVSMCs on both BAECs and the BVSMCs themselves. Incubation of BAECs with IL-1β–prestimulated BVSMCs induced EC toxicity, which was partially inhibited by an inhibitor of NO synthesis, N^ωnitro-l-arginine methyl ester, or an inhibitor of iNOS expression, dexamethasone. No cytotoxic effect of IL-1β on BVSMCs themselves was detected. ECs modulate iNOS expression in SMCs by mechanisms that include a TGF-β–dependent pathway. The NO released from SMCs exerts cytotoxic effects on the adjacent endothelium without altering the viability of the SMCs.