Experiments addressed the hypothesis that afferent and efferent arterioles differentially rely on Ca²⁺ influx and/or release from intracellular stores in generating contractile responses to AVP. The effect of Ca²⁺ store depletion or voltage-gated Ca²⁺ channel (VGCC) blockade on contractile responsiveness to AVP (0.01-1.0 nM) was assessed in blood-perfused juxtamedullary nephrons from rat kidney. Depletion of intracellular Ca²⁺ stores by 100 µM cyclopiazonic acid (CPA) or 1 µM thapsigargin treatment increased afferent arteriolar baseline diameter by 14% and 21%, respectively, but did not significantly alter efferent arteriolar diameter. CPA attenuated the contractile response to 1.0 nM AVP by 34% and 55% in afferent and efferent arterioles, respectively (P = 0.013). The impact of thapsigargin on AVP-induced afferent arteriolar contraction (52% inhibition) was also less than its effect on the efferent arteriolar response (88% inhibition; P = 0.046). In experiments probing the role of the Ca²⁺ influx through VGCCs, 10 µM diltiazem evoked a 34% increase in baseline afferent arteriolar diameter and attenuated the contractile response to 1.0 nM AVP by 45%, without significantly altering efferent arteriolar baseline diameter or responsiveness to AVP. Combined treatment with both diltiazem and thapsigargin prevented AVP-induced contraction of both vascular segments. We conclude that Ca²⁺ release from the intracellular stores contributes to the contractile response to AVP in both afferent and efferent arterioles, but is more prominent in the efferent arteriole. Moreover, the VGCC contribution to AVP-induced renal arteriolar contraction resides primarily in the afferent arteriole.