Inositol trisphosphate (IP3) formed by phospholipase C-mediated breakdown of triphosphoinositide (PIP2) may be a ubiquitous second messenger for a number of Ca2+-mobilizing receptor agonists. Using [3H]inositol-labeled rabbit peritoneal neutrophils, we report that radiolabeled inositol phosphates are generated in response to the chemotactic peptide, formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe). fMet-Leu-Phe-stimulated formation of [3H]IP3 occurs with a rapid time course and a concentration dependence which closely parallels that of stimulated lysosomal enzyme secretion. The synthetic peptide methionyl-leucyl-phenylalanine, which is unable to promote secretion, failed to elevate [3H]IP3 accumulation, and the competitive antagonist t-butyloxycarbonyl-Phe-Leu-Phe-Leu-Phe depressed the stimulant action of fMet-Leu-Phe on [3H]IP3 levels and secretion. The Ca2+ ionophore ionomycin, which promotes secretion, was unable to enhance IP3 levels, confirming that polyphosphoinositide hydrolysis is a specific receptor-mediated event that precedes calcium mobilization during neutrophil activation. The ability of leukotriene B4 to also promote a rapid accumulation of [3H]IP3 suggests that there exists in the neutrophil an interaction between phospholipase A2 and C-mediated events. These findings support the hypothesis that IP3 may be a pivotal messenger for signal transfer by Ca2+-mobilizing receptor agonists.