Atherosclerotic lesions contain monocytes/macrophages and vascular smooth muscle cells and thus may have an increased capacity for generation of nitric oxide by inducible nitric oxide synthase (NOS). We used three approaches (contractile responses, generation of L-citrulline from L-arginine, and staining with NADPH-diaphorase) to test the hypothesis that after administration of lipopolysaccharide (LPS) in vivo, generation of nitric oxide by inducible NOS is augmented in atherosclerotic arteries. New Zealand White (normal, n = 18) and Watanabe heritable hyperlipidemic (atherosclerotic, n = 21) rabbits were anesthetized and injected intravenously with vehicle or LPS. Contractile responsiveness of aortic segments was examined in vitro 4 hours after injection of LPS in vivo. There was a substantial (approximately fivefold) decrease in contractile sensitivity of aortas from LPS-treated atherosclerotic rabbits and a small (approximately twofold) decrease in normal rabbits. Incubation of aortic segments with aminoguanidine, which inhibits inducible NOS, restored contractile responsiveness after LPS treatment. In vitro assay of conversion of [14C]L-arginine to [14C]L-citrulline by aortic segments demonstrated marked (approximately fivefold) increase in calcium-independent conversion of [14C]L-arginine by LPS-treated atherosclerotic, but not normal, aortas. NADPH-diaphorase staining demonstrated positive cells only in the endothelium of normal rabbits and in the lesions and media of the atherosclerotic aortas in both vehicle- and LPS-treated rabbits. The general distribution of these NADPH-diaphorase-positive cells resembled that of smooth muscle cells and not macrophages. Thus, impairment of contractile responses, generation of L-citrulline, and staining with NADPH-diaphorase suggest that atherosclerotic arteries have increased capacity for generation of nitric oxide by inducible NOS.