CTLs kill virus-infected, tumor, and transplanted targets via secretion of lytic agents including perforin and granzymes. Knowledge of the signals controlling this important process remains vague. We have tested the idea that protein kinase C (PKC)theta, a member of the novel PKC (nPKC) family, which has been shown to play a preferential role in critical Th cell functions, plays a similar, preferential role in CTL lytic granule exocytosis using T acute lymphoblastic leukemia-104 (TALL-104) human leukemic CTLs as a model. We provide evidence consistent with the idea that PKC activity is important for the degranulation step of lytic granule exocytosis, as opposed to upstream events. In contrast with previous work, our results with pharmacological agents suggest that conventional PKCs (cPKCs) and nPKCs may participate. Our results suggest that stimulation with soluble agents that bypass the TCR and trigger granule exocytosis activates PKCalpha and PKCtheta, which can both accumulate at the site of contact with a target cell, although PKCtheta did so more often. Finally, using a novel assay that detects granule exocytosis specifically in transfected, viable cells, we find that overexpression of constitutively active mutants of PKCalpha or PKCtheta can synergize with increases in intracellular [Ca(2+)] to promote granule exocytosis. Taken together, our results lend support for the idea that PKCtheta does not play a preferential role in CTL granule exocytosis.