Since NO production is dependent on the availability of L-arginie, we examined whether L-arginine transport and NO synthesis are coregulated by vascular smooth muscle cells and endothelial cells cultured from the same vessel wall source. L-Arginine transport by both bovine aortic smooth muscle cells (BASMCs) and endothelial cells (BAECs) was primarily Na+ independent (approximately 70%) and was mediated by both a high- and low-affinity transport system. Treatment of BASMCs with tumor necrosis factor-alpha (TNF-alpha) or interleukin-1 beta (IL-1 beta) resulted in a significant increase in L-arginine transport (approximately 20%) and in the induction of NO release. Exposure of BASMCs to interferon gamma (IFN-gamma) or lipopolysaccharide (LPS) also stimulated NO release but did not affect L-arginine transport. In contrast, incubation of BAECs with TNF-alpha or LPS strikingly enhanced L-arginine uptake (2.5-fold), whereas IL-1 beta and IFN-gamma had no effect. Treatment of BAECs with any of the inflammatory mediators did not stimulate NO production. These results demonstrate that L-arginine uptake and NO synthesis by these cells are differentially regulated. In BASMCs, the coinduction of L-arginine transport and NO formation may function to provide increased levels of substrate to the cell during activation of the NO synthase enzyme. In contrast, the selective stimulation of L-arginine uptake in BAECs indicates that L-arginine transport is dissociated from NO generation in these cells.