We examined responses of renal interstitial guanosine 3',5'-cyclic monophosphate (cGMP) to changes in renal perfusion pressure (RPP) within and below the range of renal blood flow (RBF) autoregulation. A microdialysis method was used to monitor renal cortical and medullary interstitial cGMP levels in anesthetized rabbits. RPP was reduced in two steps: from 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 · min¹ · g¹. Basal interstitial concentrations of cGMP were significantly lower in the cortex than in the medulla (12.1 ± 1.4 and 19.9 ± 0.4 nmol/l, respectively). Cortical and medullary cGMP did not change in step 1 but were significantly decreased in step 2, with significantly less reduction in cGMP concentrations in the medulla than in the cortex (25 ± 3 and 44 ± 3%, respectively). Over this pressure range, changes in cortical and medullary cGMP were highly correlated with changes in RBF (r = 0.94, P < 0.005 for cortex; r = 0.82, P < 0.01 for medulla). Renal interstitial nitrate/nitrite was not changed in step 1 but was significantly decreased in step 2 (38 ± 2% in cortex and 20 ± 2% in medulla). Nitric oxide synthase inhibition with N^G-nitro-L-arginine methyl ester (L-NAME, 30 mg/kg bolus, 50 mg · kg¹ · h¹ iv infusion) significantly decreased RBF (by 46 ± 4%) and interstitial concentrations of cGMP (27 ± 4% in cortex and 22 ± 4% in medulla, respectively). 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. These results indicate that acute changes in RBF result in alterations in nitric oxide-dependent renal interstitial cGMP levels, with differential effects in the medulla compared with the cortex.