The bioluminescent oxygen metabolite indicator protein pholasin was characterized with respect to the type and location of reactive oxygen metabolites detected in suspensions of stimulated human neutrophils. Whereas pholasin detected reactive oxygen metabolites from neutrophil suspensions stimulated with soluble agents, particulate stimulants were apparently not effective triggering agents for pholasin-dependent neutrophil chemiluminescence. Neutrophils stimulated with fMet-Leu-Phe (1 to 100 nmol/l) showed maximum pholasin-dependent chemiluminescence 45 to 60 s after stimulation. The time of maximum chemiluminescence was virtually independent of fMet-Leu-Phe concentration. In contrast, the time to reach maximum light emission increased from 60 s with 100 nmol/l phorbol ester to 295 s with 1 nmol/l phorbol ester. Significant inhibition of stimulated chemiluminescence was caused by both superoxide dismutase (20 micrograms/ml, 80% inhibition) and reduction of the oxygen concentration in the incubation medium to less than 0.5 mumol/l (95% inhibition). In contrast, the myeloperoxidase inhibitor sodium azide (0.1 mmol/l) afforded only 50% inhibition of the pholasin-dependent neutrophil chemiluminescence. Our results show that pholasin detects superoxide radicals released from cells stimulated by soluble stimulants but not intracellular oxidative activity elicited by particulate stimulants.