Growth factor-induced activation of Akt (protein kinase B) is implicated in the proliferation of vascular smooth muscle cells (VSMC) in addition to antiapoptotic signaling Although previous studies have documented increases in total Akt or Akt-1 activity in rodent VSMC, little is known about the regulation of Akt-2 or Akt-3 kinase activity in VSMC from any species. In the present study, reverse transcriptase-polymerase chain reaction revealed the expression of all three Akt isoforms in human aortic VSMC. In vitro kinase assays using immunoprecipitated Akt isoforms showed robust increases in Akt-3 activity after stimulation of human aortic VSMC with platelet-derived growth factor (PDGF), insulin, and insulin-like growth factor-1. In contrast, these growth factors produced modest and marginal increases in Akt-1 and Akt-2 kinase activity, respectively. Pretreatment of VSMC with a phosphoinositide-3-kinase (PI-3K) inhibitor, LY294002, led to significant inhibition of growth factor(s)-induced increases in Akt-3 activity and DNA synthesis. The present findings provide the first direct evidence that the Akt-3 isoform is predominantly activated in human aortic VSMC. Moreover, these data suggest that PI-3K-dependent activation of Akt-3 may play a major role in VSMC proliferation.