Previous studies demonstrated a requirement for multifunctional Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) in PDGF-stimulated vascular smooth muscle (VSM) cell migration. In the present study, molecular approaches were used specifically to assess the role of the predominant CaMKII isoform (δ₂ or δ_C) on VSM cell migration. Kinase-negative (K43A) and constitutively active (T287D) mutant forms of CaMKIIδ₂ were expressed using recombinant adenoviruses. CaMKII activities were evaluated in vitro by using a peptide substrate and in intact cells by assessing the phosphorylation of overexpressed phospholamban on Thr¹⁷, a CaMKII-selective phosphorylation site. Expression of kinase-negative CaMKIIδ₂ inhibited substrate phosphorylation both in vitro and in the intact cell, indicating a dominant-negative function with respect to exogenous substrate. However, overexpression of the kinase-negative mutant failed to inhibit endogenous CaMKIIδ₂ autophosphorylation on Thr²⁸⁷ after activation of cells with ionomycin, and in fact, these subunits served as a substrate for the endogenous kinase. Constitutively active CaMKIIδ₂ phosphorylated substrate in vitro without added Ca²⁺/calmodulin and in the intact cell without added Ca²⁺-dependent stimuli, but it inhibited autophosphorylation of endogenous CaMKIIδ₂ on Thr²⁸⁷. Basal and PDGF-stimulated cell migration was significantly enhanced in cells expressing kinase-negative CaMKIIδ₂, an effect opposite that of KN-93, a chemical inhibitor of CaMKII activation. Expression of the constitutively active CaMKIIδ₂ mutant inhibited PDGF-stimulated cell migration. These studies point to a role for the CaMKIIδ₂ isoform in regulating VSM cell migration. An inclusive interpretation of results using both pharmacological and molecular approaches raises the hypothesis that CaMKIIδ₂ autophosphorylation may play an important role in PDGF-stimulated VSM cell migration.