Angiotensin II (Ang II) stimulates phospholipase D (PLD) activity and growth of vascular smooth muscle cells (VSMC). The atypical protein kinase C (PKC) 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 PKC and PLD and their implication in VSMC adhesion, spreading and hypertrophy. Ang II stimulated PKC activity with maximal activation at 30 sec followed by a decline in its activity to 45% above basal at 5 min. Inhibition of PKC activity with a myristoylated pseudosubstrate peptide or overexpression of a kinase-inactive form of PKC decreased Ang II-induced PLD activity. Moreover, depletion of PKC with selective antisense oligonucleotides also decreased Ang II-induced PLD activity. Interaction between PLD2 and PKC in VSMC was detected by co-immunoprecipitation. Ang II-induced PLD activity was inhibited by the primary alcohol n-butanol, but not the tertiary alcohol, t-butanol. The functional significance of PKC and PLD2 in VSMC adhesion, spreading and hypertrophy was investigated. Inhibition of PKC 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 PKC and suggest a crucial role of PKC-dependent PLD2 in VSMC functions such as adhesion, spreading and hypertrophy, which are associated with the pathogenesis of atherosclerosis and malignant hypertension.