The source of superoxide (O-2(.-)) production and cell-to-cell interactions of O-2(.-) and nitric oxide (NO) in response to angiotensin II (AngII) were studied by fluorescence microscopic techniques to image rat renal outer medullary microtissue strips. Changes in intracellular O-2(.-) were determined by dihydroethidium-ethidium ratios, and NO was determined with 4,5-diaminofluorescein diacetate. AngII (1 mumol/L) significantly increased O-2(.-) in the isolated, medullary thick ascending limb (mTAL). These responses were inhibited by the superoxide dismutase mimetic 4-hydroxytetramethylpiperidine-1-oxyl (TEMPOL) and by the NAD(P)H oxidase inhibitors diphenylene iodonium and apocynin. AngII did not increase O-2(.-) in either pericytes of isolated, intact vasa recta (VR) or pericytes of VR with a disrupted endothelium, even when surrounded by mTAL. However, AngII did increase O-2(.-) when the tissue strips were preincubated with the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO), indicating that cross-talk of O-2(.-) from mTAL to the VR occurred but was normally inhibited by NO. Also, tissue O-2(.-) reduction by TEMPOL increased the diffusion of NO from mTAL to the pericytes, indicating that cross-talk of NO from the mTAL to the VR is also inhibited by O-2(.-). We conclude that AngII stimulates O-2(.-) production in mTAL via the NAD(P) H oxidase pathway and that interactions of O-2(.-) and NO ultimately determine the effectiveness of in situ free-radical cross-talk between the mTAL and the VR.