We investigated the role of calcium in the activation of neutrophils (PMN) by the human lymphokine leukocyte inhibitory factor (LIF). Using the fluorescent probe fura 2, we demonstrated that LIF (1/2-2 units) induced a small but significant dose-dependent increase in intracellular calcium in the presence of both calcium-containing (71 nM to 158 nM) and calcium-free (53 nM to 144 nM) buffer solutions. Thus, LIF is able to release calcium from both membrane channels and from intracellular stores. However, increases in intracellular calcium were not due to a release of Ca+2 from membrane stores, as shown by the inability of LIF to diminish fluorescence of the membrane-bound calcium probe chlortetracycline. In contrast to formyl-methionyl-leucyl-phenylalanine (fMLP), maximal calcium fluxes caused by LIF were of a much lower magnitude and not observed until 10-15 min after its application. The importance of calcium to the metabolic effects of LIF was demonstrated by the abilities of the calmodulin inhibitor trifluoroperazine (51.5% inhibition) and the extracellular calcium chelator EGTA (50.5% inhibition) to suppress LIF-mediated degranulation. The intracellular calcium chelator fura 2 also significantly inhibited LIF-mediated degranulation (61.8% inhibition). However, inhibition of the release of calcium from intracellular stores by TMB-8, had no effect on LIF-mediated degranulation. These data suggest that the ability of LIF to activate PMN is dependent on the availability of intracellular calcium and that this calcium is primarily derived from the slow influx of calcium from the extracellular pool.