We demonstrate here that T cell receptor for antigen (TCR)-triggered exocytosis in cytotoxic T lymphocytes (CTL) is not constitutive and is regulated through crosslinking of the TCR by antigen or monoclonal anti-TCR antibodies. Morphological and biochemical data using three different biochemical markers of granules and Percoll gradient fractionation analysis are presented, suggesting that TCR-triggered exocytosis is accompanied by the loss of granules from CTL and appearance of intragranular proteins and enzymatic activities in the incubation medium. The strict requirement for crosslinking of the TCR in exocytosis triggering could be bypassed by protein kinase C activators (phorbol esters or bryostatin I and II) acting in synergy with Ca2+ ionophores. It is shown that external Ca2+ is obligatory for both the TCR-triggered and for the PMA/A23187-triggered exocytosis, since Ca2+ chelators and divalent cations that compete with Ca2+ for A23187 can inhibit exocytosis of granules. These data suggest that Ca2+ from intracellular stores is not sufficient to support exocytosis in CTL. Ca2+ channel blockers and calmodulin antagonists significantly inhibited TCR-triggered exocytosis without affecting the basal level of secretion. The described results are consistent with a model in which exocytosis of granules in CTL is triggered by the crosslinking of TCR, transmembrane protein kinase C activation, and external Ca2+ translocation through CTL plasma membrane Ca2+ channels and modulation of activity of Ca2+, calmodulin-dependent enzymes, and cytoskeletal proteins.