The effect of ampicillin on cytochrome c reduction and on the superoxide production of human neutrophils stimulated by phorbol myristate acetate (PMA) was investigated. 2. Ampicillin did not stimulate the superoxide production of intact (resting) neutrophils and not amplify the superoxide production of neutrophils stimulated by phorbol myristate acetate (PMA). 3. However, ampicillin dose-dependently increased the reduction of cytochrome c. 4. In addition, 50 mM ampicillin stimulated a superoxide dismutase-inhibitable reduction of cytochrome c by 0.70 +/- 0.02 (mean +/- SD) nmol/min and a superoxide dismutase-noninhibitable reduction of cytochrome c by 2.08 +/- 0.03 (mean +/- SD) nmol/min. 5. These results suggest that ampicillin serves as an electron donor and/or a superoxide generator.