Biochemical events that follow the engagement of cytotoxic T lymphocytes (CTL) with an Ag-bearing target cell (TC) or triggering by the crosslinking of the Ag-receptor (TcR) by immobilized anti-TcR mAb were studied using cloned CTL and a novel CTL activation assay. The approach described here was undertaken to shed light on the molecular mechanisms of" ON"," STOP" and" OFF" signalling that allow CTL to be activated, kill TC and disengage from the target cell after delivery of the" lethal hit" and then to proceed with the destruction of the next Ag-bearing target encountered. Biochemical studies of TcR-regulated and TcR-triggered constitutive exocytosis in CTL provided a detailed description of the molecular requirements for this important phenomenon in T lymphocytes and provided an alternative CTL activation assay; this assay measures the TcR-dependent response in the absence of a TC. These studies also helped to envision CTLs screening activities as a cycle of engagements-disengagements with the TC, where every surrounding cell is treated by the CTL as a potential Ag-bearing TC. Both constitutive and regulated exocytosis in CTL are triggered through a transmembrane signalling pathway which involves protein kinase C and extracellular Ca2+ that, most likely, is translocated through Ca2+ channels. This is followed by the involvement of calmodulin (CaM)-binding proteins, eg, calcineurin, a CaM-dependent phosphatase, which was shown to be a major CaM-binding protein in murine lymphocytes. Unexpectedly, these biochemical studies demonstrated that the granule exocytosis model of CTL-mediated cytotoxicity cannot account for the mechanism of target cell lysis by CTL, at least in in vitro conditions in the absence of extracellular Ca2+. These results indicate the existence of an extracellular Ca2+-independent, TcR-regulated CTL response and raise the possibility that second messenger (s) other than Ca2+ and/or products of phosphoinositide turnover are involved in T-cell lysis. Predominance of" non-lethal" engagements between some CTL and TC, revealed during time-lapse cinematographic studies, together with comparative studies of TcR-regulated exocytosis of granules and of constitutive exocytosis of gamma-interferon, suggested that TC destruction by CTL may not be their only or even their most important function in vivo. It is possible that CTL, triggered by Ag recognition to exocytose storage granules and to synthesize and constitutively exocytose macrophage-activating factors, in turn promote tumor destruction by macrophages.(ABSTRACT TRUNCATED AT 400 WORDS)