Based on evidence that extracellular ATP can influence vascular smooth muscle function in other organ systems, experiments were conducted to characterize the responsiveness of rat juxtamedullary microvascular segments to ATP. Experiments were performed using the in vitro blood-perfused juxtamedullary nephron preparation combined with video microscopy. Pentobarbital-anesthetized rats were pretreated with enalaprilat (2 mg iv) for 30 min before the right kidney was isolated and prepared for study. Renal perfusion pressure was set at 110 mmHg and held constant. Under control conditions, afferent and efferent arteriolar diameters averaged 19.9 ± 1.4 (n = 19) and 21.6 ± 1.2 μm (n = 10), respectively. Superfusion with 1, 10, and 100 μM ATP solutions induced sustained dose-dependent afferent vasoconstriction of 8.3 ± 1.4, 12.8 ± 1.7, and 12.1 ± 2.1%, respectively (P < 0.01). Afferent vasoconstrictor responses to ATP were also observed during adenosine receptor blockade. In contrast, efferent arterioles were unresponsive to ATP stimulation even at concentrations as high as 100 μM (P > 0.05). Arcuate and interlobular arterial diameters averaged 82.0 ± 15.7 (n = 5) and 43.4 ± 4.5 μm (n = 6), respectively, during control conditions and responded to ATP treatment with a transient vasoconstriction followed by a gradual return to control diameter. Interlobular arteries exhibited a sustained constriction only at the 100 μM concentration (P < 0.05). These data demonstrate that afferent arterioles are more responsive to ATP treatment than other renal microvascular segments and suggest the presence of ATP-sensitive P(2x) purinoceptors on pre- but not postglomerular juxtamedullary microvascular elements.