In the present study mechanisms of signal transduction were investigated by treatment of NCC with phorbol esters and calcium ionophore. NCC were stimulated to produce 3-fold increased cytotoxicity by 10(-4) to 10(-5) M A23187. PMA, PDD, and 4-beta phorbol either suppressed NCC lysis (PMA) or had no enhancing effects (PDD and 4-beta). At a concentration of 10(-4) M increased cytotoxicity occurred after 0.5 to 3 hours treatment. Four-hour preincubation enhanced cytotoxicity at suboptimal A23187 concentrations. A23187 produced increased NCC activity at each E:T ratio tested, however, greatest differences in A23187 concentrations were observed at 20:1 and 40:1. The effects of simultaneous treatment of NCC and/or sequential with PMA and A23187 indicated that different transduction pathways might operate to augment cytotoxicity. PMA, 10(-8) M, combined with 10(-4) M concentrations of A23187 produced a 164.7% increase in cytotoxicity compared to media controls, and a 37.7% increase compared to A23187 controls. These data indicate that teleost cytotoxic cells, unlike mammalian NK, are augmentable by increased levels of extracellular calcium without the presence of any other comodulating signals.