Exocytosis of cytotoxic granule contents towards bound target cells is thought to be of central importance in natural killer (NK) cell-mediated killing. Although cellular cytotoxicity involving degranulation is thought to be calcium-dependent, the biochemical mechanisms that mediate this granule mobilization are unknown. Inositol-1,4,5-tris-phosphate (IP3), which acts to elevate internal calcium levels, and 1,2-diacylglycerol (DAG), which activates protein kinase C, are potent second messengers that have been shown to synergistically mediate secretion in other cell types. Production of these products of inositol phospholipid metabolism has previously been demonstrated in a rat NK cell line RNK upon exposure to susceptible tumor targets. We therefore investigated the role of IP3 and DAG in NK-mediated cytotoxicity, specifically at the level of degranulation. Pretreatment of RNK cells with neomycin, a drug that interferes with the hydrolysis of inositol phospholipids and thus inhibits the formation of second messengers, inhibited RNK cytotoxicity against a susceptible tumor target and also inhibited RNK production of DAG in response to a similar target. Natural killing exhibited by normal rat nylon wool-nonadherent splenocytes was also inhibited by neomycin. Phorbol-12-myristate, 13-acetate (PMA), a phorbol ester that acts like DAG to activate protein kinase C, markedly enhanced lysis of a susceptible target cell by RNK. We evaluated whether modulation of lysis by these drugs was associated with effects on RNK degranulation by assaying the release of a granule-specific serine esterase (BLTE) in response to PMA and the calcium ionophore A23187. These agents synergized to promote the release of BLTE, and the extent of release was dependent on the concentrations of both agents. D2O and cytochalasin B, which enhance secretion in other cells, both enhanced BLTE release from RNK cells, indicating that we were detecting BLTE released via granule secretion and not due to nonspecific causes such as cell lysis. Our findings lead us to propose that NK cells form IP3 and DAG in response to susceptible target cells and that a major function of these second messengers is to mediate the exocytosis of cytotoxic granules towards the bound target cells.