Angiotensin II (Ang II) promotes neointimal growth in the balloon-injured rat carotid artery. However, the mechanism by which Ang II stimulates neointimal growth during vascular injury is not known. In cultured vascular smooth muscle cells (VSMC), Ang II activates Akt through cytosolic phospholipase A₂ (cPLA₂)-dependent phospholipase D₂ (PLD₂). This study was conducted to determine if Ang II-induced neointimal thickening is mediated via cPLA₂ and PLD₂ -activated Akt in balloon-injured rat carotid arteries. Ang II- stimulated neointimal growth was inhibited by exposure of the injured carotid arteries to an adenovirus containing a dominant negative (DN) Akt mutant (intima/media ratio: from 3.01±0.31 to 1.44±0.14, P<0.01) or a retrovirus containing cPLA₂ siRNA (intima/media ratio: from 3.01±0.31 to 1.16±0.36, P<0.001) or PLD₂ small interfering RNA (siRNA) (intima/media ratio: from 3.01±0.31 to 1.33±0.11, P<0.001). The effect of cPLA₂ and PLD₂ siRNA to reduce the Ang II-induced increase in neointimal thickening was associated with reduced expression of cPLA₂ and PLD₂, as determined by immunohistochemical analysis in injured carotid arteries. Western blot analysis showed that Akt phosphorylation that was increased by Ang II was inhibited in injured carotid arteries two days after exposure to cPLA₂ or PLD₂ siRNA or in injured arteries isolated after exposure to these agents for 30 min and then placed in tissue culture media for 24 h in the presence of these agents. These data suggest that the Ang II-induced neointimal growth is mediated by activation of Akt through a mechanism dependent upon cPLA₂ and PLD₂ activation in balloon-injured rat carotid arteries.