Metabolic pathways involved in the activation of polymorphonuclear leukocytes (PMNs) were characterized by using chemoattractants with equivalent chemotactic activity but widely disparate ability to stimulate superoxide production [N-formylmethionylleucylphenylalanine (fMet-Leu-Phe) much greater than leukotriene B4]. Leukotriene B4 stimulated a low level of superoxide production that plateaued at 60 sec, whereas with fMet-Leu-Phe the response continued to increase for 5 min. Both agents produced equivalent initial rises in diacylglycerol (acyl2Gro) (less than or equal to 30 sec); however, only fMet-Leu-Phe induced a second increase of acyl2Gro peaking at ca. 120 sec. Both chemoattractants also caused an equivalent initial (less than or equal to 10 sec) rise in intracellular calcium; however, the elevation induced by fMet-Leu-Phe was more sustained. We sought to determine the biochemical mechanisms underlying these discrepancies. Superoxide production and the second phase of acyl2Gro generation were both inhibited ca. 56% by depleting extracellular calcium or ca. 79% by buffering intracellular calcium. Cytochalasin B greatly enhanced the respiratory burst, acyl2Gro production, and calcium influx, but not inositolphospholipid turnover in PMNs stimulated with chemoattractants. These data indicate that sequential metabolic pathways activate the respiratory burst in PMNs stimulated by chemoattractants. The response is initiated by inositolpolyphospholipid hydrolysis, which results in rapid (less than or equal to 5 sec) calcium mobilization from intracellular stores and acyl2Gro release (peak at ca. 30 sec). To fully activate the respiratory burst, the chemoattractant must also trigger calcium influx, which leads to a sustained cytosolic calcium elevation. This supports a prolonged new phase of acyl2Gro production that is independent of inositolphospholipid hydrolysis and is correlated with superoxide production.