We recently demonstrated a direct relationship between autoregulation-related changes in renal vascular resistance (RVR) and renal interstitial ATP concentrations. To assess the possible role for extracellular ATP in the regulation of tubuloglomerular feedback (TGF)-mediated autoregulatory adjustments in RVR, renal interstitial ATP concentrations were measured with microdialysis probes in anesthetized dogs at different renal arterial pressures (RAPs) within the autoregulatory range during augmented and diminished activity of the TGF mechanism. Stepwise reductions in RAP from ambient pressure (129±3 mm Hg) to 102±2 mm Hg (step 1) and 75±1 mm Hg (step 2) resulted in significant decreases in ATP concentrations from 9.0±0.8 to 6.3±0.6 nmol/L in step 1 and to 4.2±0.5 nmol/L in step 2. Changes in RVR were highly correlated with changes in ATP concentrations (r=0.86, P<0.001, n=12). Acetazolamide (100 μg · kg⁻¹ · min⁻¹, n=6), which increases solute delivery to the macula densa, thus augmenting TGF activity, significantly decreased renal blood flow (RBF) by −16±2% and glomerular filtration rate (GFR) by −22±4% and increased ATP concentrations from 8.4±0.7 to 15.5±1.4 nmol/L. Although basal RBF and GFR levels were reduced by the acetazolamide infusion, autoregulation efficiency was maintained, and interstitial ATP concentrations were significantly decreased in response to reductions in RAP by −36±4% in step 1 and by −54±2% in step 2. The relationship between changes in RVR and interstitial ATP concentrations was preserved during acetazolamide treatment (r=0.80, P<0.01). Inhibition of the TGF mechanism by furosemide significantly increased RBF by 33±6% and GFR by 13±2% and decreased ATP concentrations from 8.9±1.4 to 5.0±0.8 nmol/L (n=6). Furosemide caused marked impairment of RBF and GFR autoregulatory efficiency (by −14±3% and −11±3% in step 1 and by −26±2% and −18±4% in step 2, respectively). In the furosemide-treated kidneys, interstitial ATP levels remained low and were not altered during reductions in RAP (4.7±0.7 nmol/L in step 1 and 4.7±0.8 nmol/L in step 2), and changes in RVR did not exhibit a correlation with changes in ATP concentrations (r=0.22, P=0.30). These data support the hypothesis that extracellular ATP contributes to autoregulatory adjustments in RVR that are mediated by changes in activity of the TGF mechanism.