The protein kinase C (PKC) Ser/Thr kinases account for approximately 2% of the human kinome and regulate diverse cellular behaviors. PKC catalytic activity requires priming phosphorylations at three conserved sites within the kinase domain. Here we demonstrate that priming of PKC is dependent on the conformation of the nucleotide binding pocket but not on its intrinsic kinase activity. Inactive ATP binding site mutants are unprimed, but they become phosphorylated upon occupancy of the ATP binding pocket with inhibitors of PKC. We have exploited this property to screen for PKC inhibitors in vivo. Further, we generated a distinct class of kinase-inactive mutants that maintain the integrity of the ATP binding pocket; such mutants are constitutively primed and functionally distinct from ATP binding site mutants. These data demonstrate that autophosphorylation is not required for PKC priming and show how ATP pocket occupation can enable a kinase to mature as well as function.