Nitric oxide (NO) plays an important role in the cytotoxic mechanisms responsible for acute renal allograft rejection, where macrophages produce high levels of inducible nitric oxide synthase (iNOS). By contrast, both the source and the role of NO in chronic allograft nephropathy (CAN) are still unclear. In this study, the expression of iNOS mRNA and protein was assessed in the kidneys of patients with graft failure due to chronic rejection. As controls, kidney specimens were obtained from patients undergoing nephrectomies for primary renal tumours, and from patients suffering from IgA nephropathy or mesangial-proliferative glomerulonephritis. In normal kidneys, iNOS production was absent or limited to a low signal, while it was found only in the inflammatory infiltrate of kidneys affected by glomerulonephritis, as assessed by immunohistochemistry and in situ hybridization. In contrast, in CAN, iNOS protein was localized not only in inflammatory cells, but also in vascular, glomerular, and, more rarely, tubular structures. Accordingly, in situ hybridization localized iNOS mRNA in both macrophages and lymphocytes, as well as in vascular structures and glomeruli. Double immunostaining for iNOS and a-smooth muscle actin (a-SMA) or von Willebrand factor (vWf) revealed that smooth muscle cells were the main vascular source of iNOS, while both mesangial and inflammatory cells were immunostained at the glomerular level. These data demonstrate that macrophages and lymphocytes are not the only source of iNOS mRNA and protein in human CAN. Vascular smooth muscle and mesangial cells also synthesize iNOS, raising the question of heterogeneous regulation and function of iNOS in this disease.