The signaling output of protein kinase C (PKC) is exquisitely controlled, with its disruption resulting in pathophysiologies. Identifying the structural basis for autoinhibition is central to developing effective therapies for cancer, where PKC activity needs to be enhanced, or neurodegenerative diseases, where PKC activity should be inhibited. Here, we reinterpret a previously reported crystal structure of PKCbetaII and use docking and functional analysis to propose an alternative structure that is consistent with previous literature on PKC regulation. Mutagenesis of predicted contact residues establishes that the Ca(2+)-sensing C2 domain interacts intramolecularly with the kinase domain and the carboxyl-terminal tail, locking PKC in an inactive conformation. Ca(2+)-dependent bridging of the C2 domain to membranes provides the first step in activating PKC via conformational selection. Although the placement of the C1 domains remains to be determined, elucidation of the structural basis for autoinhibition of PKCbetaII unveils a unique direction for therapeutically targeting PKC.