Previous studies have suggested a paracrine role for extracellular ATP in the regulation of afferent arteriolar tone. The current study was conducted to determine the dependence of this response on calcium entry mechanisms. Experiments were performed in vitro using the blood-perfused juxtamedullary nephron technique combined with video microscopy. The afferent arteriolar response to α,β-methylene ATP was determined before and after treatment with the calcium channel blockers, diltiazem or felodipine. α,β-Methylene ATP was used to obviate concerns over responses being elicited by ATP or by ATP hydrolysis products such as adenosine. Previous studies have shown that afferent arteriolar responses to α,β-methylene ATP are comparable to those elicited by ATP. α,β-Methylene ATP (1.0 μM) induced a rapid initial afferent vasoconstriction of 72.5 ± 10.6%, which partially recovered to a stable diameter 11.3 ± 1.7% smaller than control (P < 0.01 vs. control). Afferent diameter returned to control diameter on removal of ATP from the bath. Diltiazem or felodipine treatment significantly increased afferent diameter by 5.6 ± 2.3 and 16.4 ± 4.6%, respectively (P < 0.05). In the presence of either diltiazem or felodipine, the initial vasoconstriction to α,β-methylene ATP was attenuated, and the sustained vasoconstriction was completely blocked. Removal of calcium from the extracellular medium completely abolished both the initial and sustained vasoconstrictor response. Returning the extracellular calcium concentration to the physiological range completely restored the biphasic vasoconstrictor response to α,β-methylene ATP. These data suggest that both the initial and the sustained phases of the ATP-mediated juxtamedullary afferent arteriolar vasoconstriction are dependent on the influx of extracellular calcium and that the sustained constriction is dependent primarily on calcium entry via voltage-gated L- type calcium channels.