The present study tested the hypothesis that membrane-bound NAD(P)H oxidase in coronary arterial myocytes (CAMs) is capable of producing superoxide (O₂^.-) toward extracellular space to exert an autocrine- or paracrine-like action in these cells. Using a high speed wavelength switching fluorescent microscopic imaging technique, the binding of dihydroethidium (DHE) oxidizing product to exogenous salmon testes DNA trapped outside CAMs and to nuclear DNA were simultaneously monitored as indicators of extra- and intracellular O₂^.- production. It was found that a muscarinic agonist, oxotremorine (OXO, 80 µM) increased O₂^.- levels more rapidly outside than inside CAMs. In the presence of superoxide dismutase (SOD, 500 U/ml) plus catalase (CAT, 400 U/ml) and NAD(P)H oxidase inhibitor diphenylene iodonium (DPI, 50 µM) or apocynin (APO, 100 µM), these increases in both extra- and intracellular O₂^.- levels were substantially abolished or attenuated. The O₂^.- increase outside CAMs was also confirmed by detecting oxidation of nitro blue tetrazolium (NBT) and confocal microscopic localization of Matrigel trapped OxyBURST H2HFF Green BSA staining around these cells. By electron spin resonance (ESR) spectrometry, the extracellular accumulation of O₂^.- was demonstrated as a SOD-sensitive component outside CAMs. Furthermore, RNA interference of NAD(P)H oxidase subunits NOX1 or p47 markedly blocked OXO-induced increases in both extra- and intracellular O₂^.- levels, while siRNA of NOX4 only attenuated intracellular O₂^.- accumulation. These results suggest that NAD(P)H oxidase activity contributes to OXO-induced increases in O₂^.- levels both outside and inside CAMs and that NOX1 represents a major NOX isoform responsible for extracellular O₂^.- production. This rapid extracellular production of O₂^.- seems to be unique to OXO-induced M1-receptor activation, since angiotensin II (Ang II)-induced intra- and extracellular O₂^.- increase in parallel. It is concluded that the outward production of O₂^.- via NAD(P)H oxidase in CAMs may represent an important producing pattern for its autocrine or paracrine actions.