A simple, rapid and inexpensive method for the measurement of hydrogen peroxide (H2O2) produced by cells in culture is described. The assay is based on the horseradish peroxidase (HRPO)-mediated oxidation of phenol red by H2O2 which results in the formation of a compound demonstrating increased absorbance at 610 nm. A linear relationship between absorbance at 610 nm and concentration of H2O2 was found in the 1--60 microM (1--60 nmoles/ml) range. Due to the non-toxic character of phenol red and HRPO, the assay permits measurement of H2O2 production and release by macrophages for time intervals of 5--60 min under regular tissue culture conditions. Using this assay, the ability of a number of agents to induce H2O2 release by guinea pig peritoneal macrophages was demonstrated. These agents were: phorbol myristate acetate (PMA), opsonized zymosan, concanavalin A (Con A), wheat germ agglutinin (WGA), N-formyl-methionyl-leucyl-phenylalanine (FMLP) and A23187.