Generation of superoxide anion (O2-) and mobilization of intracellular Ca2+ in human neutrophils upon exposure to stimuli such as N-formylmethionylleucylphenylalanine (fMLP) were inhibited by the antibiotic cerulenin, which inhibits fatty acid and cholesterol biosynthesis. The inhibition of O2- generation and Ca2+-mobilization required certain periods of incubation with cerulenin and both abilities of the cells were gradually lost following a similar time-course. In contrast, significant Ca2+-influx from the medium was observed in cerulenin-treated cells as well as untreated cells. The results suggest that an event which coincides with the Ca2+-mobilization and not Ca2+ per se is important for the induction of O2- generation in the fMLP-stimulated cells and that this step is blocked in cerulenin-treated cells. Phorbol myristate acetate or synthetic 1-oleoyl-2-acetylglycerol were able to bypass the block and induced O2- generation in cerulenin-treated cells.