Using a cloned murine cell line, NKB61A2, that concomitantly exhibits both NK and natural cytotoxic (NC) activities, we investigated the biochemical mechanisms involved in natural cell mediated cytotoxicity against NK-sensitive YAC-1 tumor cells and against the NC-sensitive WEHI-164 tumor cells. Recent reports have suggested that target cell lysis by cytotoxic lymphocytes occurs by either a calcium dependent and/or a calcium-independent mechanism(s). To determine the role of calcium in NK and NC activities of the NKB61A2 cell line, we evaluated the effect of: 1) extracellular Ca2+ depletion by the divalent cation chelator, EGTA, 2) Ca2+ influx blockade by the Ca2+ channel blocker verapamil, and 3) blocking of intracellular Ca2+ mobilization by 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8). We found that EGTA, verapamil, and TMB-8 were all capable of inhibiting NK activity, but they had little effect on NC activity of the NKB61A2 cells. Using 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide which are inhibitors of protein kinase C and calmodulin respectively, we determined that protein kinase C and calmodulin do play a role in the NK activity of NKB61A2 cells. 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine and N-(6-aminohexyl)-5-chloro-1-naphthalanesulfonamide, similar to Verapamil and TMB-8, had no effect on NC activity. Thus, the data indicate that the NK activity of NKB61A2 cells is calcium dependent whereas NC activity is not. These results may explain the disparate reports seen in the literature of calcium-dependent and -independent lysis of tumor cells.