Protein kinase C theta (PKCtheta) is a member of the novel or nPKC family. A functional role for PKCtheta is unknown. The present studies demonstrate that PKCtheta is cleaved in the third variable region (V3) in apoptosis induced by diverse agents. PKCtheta cleavage is blocked in cells that overexpress the anti-apoptotic Bcl-xL or the baculovirus p35 protein. PKCtheta is cleaved by Caspase-3 and by apoptotic cell lysates at a DEVD354/K site. We also show that overexpression of the cleaved kinase-active PKCtheta fragment, but not full-length PKCtheta or a kinase-inactive fragment, results in induction of sub-G1 phase DNA, nuclear fragmentation, and lethality. These findings indicate that proteolytic cleavage of PKCtheta by Caspase-3 induces events characteristic of apoptosis.