After injury to the blood vessel wall, vascular smooth muscle cells (SMC) synthesize interleukin (IL)-1 and inducible nitric oxide (NO) synthase (iNOS). The present study tested whether endogenous production of IL-1 stimulates iNOS expression in vascular SMC, and assessed whether IL-1 exerts autocrine effects on the cells producing IL-1 or juxtacrine effects on cells that contact the IL-1 producing cells. Rat aortic SMC were transiently transfected with expression plasmids encoding either IL-1 precursor, which localizes to the plasma membrane, or mature IL-1, which remains cytosolic. iNOS mRNA levels, determined by RT-PCR, and production of nitrite, a stable oxidation product of NO, were markedly elevated in SMC overexpressing IL-1 precursor, and modestly elevated in SMC overexpressing mature IL-1, relative to SMC transfected with vector alone. Exposure to exogenous IL-1 or TNF- further stimulated iNOS gene expression in SMC producing IL-1; low levels of IL-1 (20 pg/ml) were effective in SMC transfected with IL-1 precursor plasmid, whereas SMC transfected with mature IL-1 plasmid or vector alone required higher concentrations of IL-1 (200 and 2,000 pg/ml, respectively). The increases in iNOS mRNA levels and NO production in SMC overexpressing IL-1 precursor were prevented by exogenous IL-1 receptor antagonist, suggesting that these effects were mediated by the type I IL-1 receptor. Immunostaining studies indicated that IL-1 precursor stimulates iNOS gene expression via cell-cell contact. Expression of iNOS was enhanced in cells that were in contact with a cell overexpressing IL-1 precursor (identified by coexpression of green fluorescent protein), and in cells that were overexpressing IL-1 themselves, but only when the cell contacted another cell. Together these results indicate that IL-1 precursor acts by cell-cell contact as an autocrine and juxtacrine enhancer of iNOS gene expression, inducing moderate iNOS expression on its own, and markedly augmenting the responsiveness of rat aortic SMC to exogenous cytokines.