Arterial smooth muscle (ASM) contraction plays a critical role in regulating blood distribution and blood pressure. Vasoconstrictors activate cell surface receptors to initiate signaling cascades involving increased [Ca²⁺]_i and recruitment of protein kinase C (PKC), leading to ASM contraction, though the PKC isoenzymes involved vary between different vasoconstrictors and their actions. Here, we have used confocal microscopy of eGFP-labeled PKC isoenzymes to visualize PKC translocation in primary rat mesenteric ASM cells in response to physiological vasoconstrictors, with simultaneous imaging of Ca²⁺ signaling. Endothelin-1, angiotensin II and uridine triphosphate (UTP) all caused translocation of each of the PKC isoenzymes , and , however, the kinetics of translocation varied between agonists and PKC isoenzymes. Translocation of eGFP-PKC mirrored the rise in [Ca²⁺]_i, while that of eGFP-PKC or occurred more slowly. Endothelin-induced translocation of eGFP-PKC was often sustained for several minutes, while responses to angiotensin II were always transient. In addition, preventing [Ca²⁺]_i increases using BAPTA-AM prevented eGFP-PKC translocation, while eGFP-PKC translocated more rapidly. Our results suggest that PKC isoenzyme specificity of vasoconstrictor actions occurs downstream of PKC recruitment, and demonstrate the varied kinetics and complex interplay between Ca²⁺ and PKC responses to different vasoconstrictors in ASM.