ANG II stimulates phospholipase D (PLD) activity and growth of vascular smooth muscle cells (VSMC). The atypical protein kinase C-zeta (PKCzeta) plays a central role in the regulation of cell survival and proliferation. This study was conducted to determine the relationship between ANG II-induced activation of PKCzeta and PLD and their implication in VSMC adhesion, spreading, and hypertrophy. ANG II stimulated PKCzeta activity with maximal activation at 30 s followed by a decline in its activity to 45% above basal at 5 min. Inhibition of PKCzeta activity with a myristoylated pseudosubstrate peptide or overexpression of a kinase-inactive form of PKCzeta decreased ANG II-induced PLD activity. Moreover, depletion of PKCzeta with selective antisense oligonucleotides also decreased ANG II-induced PLD activity. Interaction between PLD2 and PKCzeta in VSMC was detected by coimmunoprecipitation. ANG II-induced PLD activity was inhibited by the primary alcohol n-butanol but not the tertiary alcohol t-butanol. The functional significance of PKCzeta and PLD2 in VSMC adhesion, spreading, and hypertrophy was investigated. Inhibition of PKCzeta and PLD2 activity or expression attenuated VSMC adhesion to collagen I and ANG II-induced cell spreading and hypertrophy. These results demonstrate that ANG II-induced PLD activation is regulated by PKCzeta and suggest a crucial role of PKCzeta-dependent PLD2 in VSMC functions such as adhesion, spreading, and hypertrophy, which are associated with the pathogenesis of atherosclerosis and malignant hypertension.