The inner medullary collecting duct (IMCD) contains relatively high nitric oxide (NO) synthetic capacity, but the effect of NO on IMCD transport remains unclear. We determined the effect of NO on basal and vasopressin (AVP)-stimulated urea (P_urea) and water (P_f) permeabilities in isolated-perfused rat IMCD. The NO donor S-nitroso-N-acetylpenicillamine (SNAP) increased cyclic GMP production in IMCD, but neither SNAP (10^-4 M) nor the cell permeable analogue of cyclic GMP, 8-Br-cyclic GMP (10^-4 M) affected basal or AVP-stimulated P_urea. The free radical superoxide is produced by oxidases in the kidney, and can interact with NO. To determine the effect of superoxide generation on transport, IMCD were incubated with the inhibitor of superoxide dismutase (SOD), diethyldithiocarbamate (DETC, 10^-3 M). DETC significantly increased basal and AVP-stimulated urea permeability (P_urea: control: 28.7 ± 4.5 vs. DETC: 40.9 ± 6.2 x 10^-5 cm/s; P<0.001; n=9). Preincubation of IMCD with SNAP or the SOD mimetic TEMPOL completely inhibited DETC-stimulated P_urea. DETC caused a significant increase in superoxide generation by IMCD, and this was blocked by SNAP. Incubation of IMCD with the NO synthase (NOS) substrate L-arginine blocked the stimulatory effect of DETC on P_urea, and this was reversed by the neuronal NOS inhibitor, 7-nitroindazole. In contrast, basal- or AVP-stimulated P_f were not affected by NO donors or DETC. In summary, exogenous or endogenously produced NO does not affect basal urea transport in the IMCD, but inhibits superoxide-stimulated urea permeability. In the inner medulla, superoxide generation by local oxidases may stimulate urea transport, and the role of endogenous NO may be to dampen this effect by decreasing superoxide levels.