The cell-free activation of human neutrophil NADPH oxidase (02- generating enzyme) was enhanced by exogenously added G-actin (actin monomer). When cytosol, a constituent of the system, was pretreated with DNase I, which may bind to G-actin (endogenous) to block polymerization, the activation of NADPH oxidase was significantly suppressed. The activation was also impaired when cytosol G-actin was removed by DNase I-linked resin, being completely restored by the addition of G-actin. These results suggest a role of actin and its polymerization in the activation of NADPH oxidase of human neutrophils.