Current scientific literature generally attributes the vasoconstrictor effects of [Arg⁸]-vasopressin (AVP) to the activation of phospholipase C (PLC) and consequent release of Ca²⁺ from the sarcoplasmic reticulum. However, half-maximal activation of PLC requires nanomolar concentrations of AVP, whereas vasoconstriction occurs when circulating concentrations of AVP are orders of magnitude lower. Using cultured vascular smooth muscle cells, we previously identified a novel Ca²⁺ signaling pathway activated by 10-100pM AVP. This pathway is distinguished from the PLC pathway by its dependence upon protein kinase C (PKC) and L-type voltage-sensitive Ca²⁺ channels (VSCC). In the present study, we used isolated pressurized rat mesenteric arteries to examine the contributions of these different Ca²⁺ signaling mechanisms to AVP-induced vasoconstriction. AVP (10^-14 to 10^-6 M) induced a concentration-dependent constriction of arteries that was reversible with a V_1a vasopressin receptor antagonist. Half maximal vasoconstriction at 30pM AVP was prevented by blockade of VSCC with verapamil (10µM) or by PKC inhibition with calphostin-C (250nM) or Ro-31-8220 (1µM). In contrast, acute vasoconstriction induced by 10nM AVP (maximal), was insensitive to blockade of VSCC or PKC inhibition. However, after 30 min the remaining vasoconstriction induced by 10nM AVP was partially dependent on PKC activation and almost fully dependent upon VSCC. These results suggest that different Ca²⁺ signaling mechanisms contribute to AVP-induced vasoconstriction over different ranges of AVP concentration. Vasoconstrictor actions of AVP, at concentrations of AVP found within the systemic circulation, utilize a Ca²⁺ signaling pathway that is dependent upon PKC activation, and can be inhibited by Ca²⁺ channel blockers.