Phosphorylation sites in members of the protein kinase A (PKA), PKG, and PKC kinase subfamily are conserved. Thus, the PKB kinase PDK1 may be responsible for the phosphorylation of PKC isotypes. PDK1 phosphorylated the activation Loop sites of PKC zeta and PKC delta in vitro and in a phosphoinositide 3-kinase (PI 3-kinase)-dependent manner in vivo in human embryonic kidney (293) cells. All members of the PKC family tested formed complexes with PDK1, PDK1-dependent phosphorylation of PKC delta in vitro was stimulated by combined PKC and PDK1 activators. The activation Loop phosphorylation of PKC delta in response to serum stimulation of cells was PI 3-kinase-dependent and was enhanced by PDK1 coexpression.