Phenotypic modulation of vascular myocytes is important for vascular development and adaptation. A characteristic feature of this process is alteration in intracellular Ca²⁺ handling, which is not completely understood. Here, we studied mechanisms involved in functional changes of IP₃- and ryanodine (RY)-sensitive Ca²⁺ stores, store-operated Ca²⁺ entry (SOCE) and receptor-operated Ca²⁺ entry (ROCE) associated with arterial myocyte modulation from a contractile to a proliferative phenotype in culture. Proliferating, cultured myocytes from rat mesenteric artery have elevated resting cytosolic Ca²⁺ levels and increased IP3-sensitive Ca²⁺ store content. ATP- and cyclopiazonic acid (CPA, SERCA inhibitor)-induced Ca²⁺ transients in Ca²⁺-free medium are significantly larger in proliferating arterial smooth muscle cells (ASMCs) than in freshly dissociated myocytes, whereas caffeine (CAF)-induced Ca²⁺ release is much smaller. Moreover, the CAF/RY-sensitive store gradually loses sensitivity to CAF activation during cell culture. These changes can be explained by increased expression of all three IP3 receptors and a switch from RY receptor type II to type III expression during proliferation. SOCE, activated by depletion of the IP3/CPA-sensitive store, is greatly increased in proliferating ASMCs. Augmented SOCE and ROCE (activated by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol) in proliferating myocytes can be attributed to upregulated expression of, respectively, transient receptor potential proteins, TRPC 1/4/5 and TRPC 3/6. Moreover, STIM1 and Orai proteins are upregulated in proliferating cells. Increased expression of IP₃ receptors, SERCA2b, TRPCs, Orai(s) and STIM1 in proliferating ASMCs suggests that these proteins play a critical role in an altered Ca²⁺ handling that occurs during vascular growth and remodeling.