Human neutrophils stimulated with phorbol myristate acetate (PMA) were able to damage human erythroleukemic K-562 target cells as assessed by a 3-hr 51Cr-release assay. Neutrophils from a patient with chronic granulomatous disease of childhood were ineffective in mediating PMA-stimulated cytolysis. Cytotoxicity was inhibited under anaerobic conditions as well as by catalase and several free radical scavengers. Superoxide dismutase, azide, and cyanide failed to inhibit PMA-dependent cytotoxicity. The influence of the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) on stimulus-dependent cytotoxicity was examined. Neutrophils preincubated with 1.0 X 10(-7) M FMLP did not display an increased cytotoxic response, but were markedly amplified in their ability to effect cytotoxicity with the addition of PMA. Enhancement of PMA-stimulated cytotoxicity resulted from a reversible cellular response to FMLP. The kinetics of the cytotoxic responses reflected the possibility that chemotactic peptide-treated neutrophils released cytotoxic mediators to a greater extent and at a faster rate than did untreated neutrophils. Although azide and superoxide dismutase did not inhibit cytotoxic responses of chemotactic peptide-activated neutrophils, the response was prevented by catalase and was markedly inhibited by several free radical scavengers. The ability of FMLP to enhance cytotoxic responses correlated well with its enhancement of PMA-stimulated chemiluminescence under a variety of conditions. In addition, the ability of PMA-stimulated neutrophils to mediate methane generation from dimethyl sulfoxide and ethylene generation from alpha keto-gamma-methiol-butyric acid (KMB)--assays that quantitate the generation of oxidizing radicals--was increased if the neutrophils were preincubated with FMLP. These results demonstrate that a chemotactic factor greatly potentiates the release of cytotoxic mediators from neutrophils upon stimulation with a nonchemotactic agent. The cytotoxic mediators appear to be products of oxidative metabolism. The cytotoxic potential of neutrophils that have responded to chemotactic stimuli to reach sites of inflammation may be activated in a similar manner.