We have evaluated the responses of renal interstitial cyclic guanosine 3',5'-monophosphate (cGMP) to changes in renal arterial pressure (RPP) within and below the range of renal blood flow (RBF) autoregulation. Using an in vivo microdialysis method, renal cortical and medullary interstitial cGMP levels were monitored in anesthetized rabbits. RPP was reduced in 2 steps, one from the ambient pressure (89±3 mmHg) to 70±2 mmHg (Step-1) and then to 48±3 mmHg (Step-2). RBF was maintained in Step-1 but was significantly decreased in Step-2 from 2.94±0.23 to 1.47±0.08 mL/min^-1/g^-1. The basal interstitial concentrations of cGMP and nitrate/nitrite in the cortex (12.1±1.4 nmol/L and 41±2 µmol/L, respectively) were significantly lower than those in the medulla (19.9±0.4 nmol/L and 62±5 µmol/L, respectively). Renal interstitial concentrations of cGMP in the cortex and medulla did not change in Step-1, but these levels were significantly decreased in Step-2 with the magnitude of reductions in cGMP concentrations in the medulla (-25±3%) being significantly less than those in the cortex (-44±3%). Over this pressure range, changes in cortical and medullary interstitial concentrations of cGMP were highly correlated with changes in RBF (cortex, r=0.94, P<0.005; medulla, r=0.82, P<0.01). Cortical and medullary interstitial concentrations of nitrate/nitrite were not changed in Step-1, but were significantly decreased in Step-2 (cortex, -38±2%; medulla, -20±2%). Nitric oxide (NO) synthase inhibition with N^G-nitro-L-arginine methyl ester (L-NAME: 30 mg/kg^-1, bolus and 50 mg/kg^-1/h^-1, infusion for 60-min, i.v.) significantly decreased RBF (by -46±4%) and interstitial concentrations of cGMP (cortex, -27±4%; medulla, -22±4%, respectively). Although basal RBF levels were reduced by L-NAME, equally efficient autoregulation was observed in response to reductions in RPP. During L-NAME treatment, renal interstitial concentrations of cGMP in the cortex and medulla were similarly not altered in Step-1. However, L-NAME significantly attenuated cGMP responses to a reduction in RPP in Step-2 (cortex, -20±3%; medulla, -14±3%). These results indicate that acute changes in RBF result in alterations in NO-dependent renal interstitial cGMP levels, with differential effects in the medulla compared to the cortex.