Protein kinase Bα (PKBα/Akt-1) is a key mediator of multiple signaling pathways involved in angiogenesis, cell proliferation and apoptosis among others. The unphosphorylated form of Akt-1 is virtually inactive and its full activation requires two phosphatidylinositol-3,4,5-triphosphate-dependent phosphorylation events, Thr³⁰⁸ by 3-phosphoinositide-dependent kinase-1 (PDK1) and Ser⁴⁷³ by an undefined kinase that has been termed PDK2. Recent studies have suggested that the Ser⁴⁷³ kinase is a plasma membrane raft-associated kinase. In this study we show that protein kinase Cα (PKCα) translocates to the membrane rafts in response to insulin growth factor-1 (IGF-1) stimulation. Overexpression of PKCα increases Ser⁴⁷³ phosphorylation and Akt-1 activity, while inhibition of its activity or expression decreases IGF-1-dependent activation of Akt-1. Furthermore, in vitro, in the presence of phospholipids and calcium, PKCα directly phosphorylates Akt-1 at the Ser⁴⁷³ site. We conclude, therefore, that PKCα regulates Akt-1 activity via Ser⁴⁷³ phosphorylation and may function as PDK2 in endothelial cells.