Vascular endothelial cell superoxide (O-2(.)) has an important role in intracellular signaling, in interaction with other reactive species such as nitric oxide, and in vascular dysfunction. Little is known regarding the source and function of O-2(.) from microvascular endothelial cells from specific tissues. Mouse lung microvascular endothelial cells stimulated with phorbol ester (PMA) or NADPH generated significant O-2(.), which was inhibited by diphenyleneiodonium (DPI) but not by allopurinol, rotenone, indomethacin, or quinacrine. Optimal O-2(.) generation required cytosolic as well as particulate cell fractions of cells. In parallel studies, PMA induced increased expression of the p47 component of the NAD(P)H oxidase in the particulate fraction, which was inhibited by staurosporine and calphostin. These data demonstrate that NAD(P)H oxidase is an important source of O-2(.) generation in lung microvascular endothelial cells.