Using neomycin, which inhibits phosphoinositide breakdown, cytotoxicity mediated by natural killer (NK) cells was suppressed in a dose-dependent manner, with complete inhibition at 16 mM. Generation of inositol phosphates in effector cells after target cell binding was inhibited in the presence of neomycin. The formation of effector to target cell conjugates was not affected. Neomycin-induced inhibition of NK killing was abolished when TPA was added to the cytotoxic assays. This reconstitution was dependent upon extracellular Ca2+. When the intracellular free Ca2+ level in effector cells was reduced from 73 +/- 11 nM to 43 +/- 3 nM (n = 4) using the Ca2+ indicator dye, Quin 2, NK killing was markedly reduced. Inhibiting the enzyme diacylglycerol (DG) kinase in effector cells with 10 microM R59022 (DG kinase inhibitor) potentiates NK killing, suggesting an increase in protein kinase C (PKC) activity due to accumulation of DG. The PKC inhibitor, H-7, suppressed NK killing in a concentration-dependent manner. These results demonstrate that phosphoinositide metabolism is an early event and its derived second messengers play a central role in activating the lytic mechanism of NK cells.