Phorbol 12-myristate 13-acetate induces the release of a low molecular weight clastogenic factor from monocytes. Hydroperoxy-5,8,11,13-icosatetraenoic acids represent major components of clastogenic factor. We report that several isomeric hydroperoxy-5,8,11,13-icosatetraenoic acids efficiently induce DNA strand breakage and/or alkali-labile sites in the mouse embryo fibroblasts C3H/10T1/2. Fe chelation by desferrioxamine suppresses breakage by approximately equal to 42% indicating the participation of Fe-catalyzed radical reactions. An additional 37% inhibition is observed upon addition of the Ca2+ chelators EGTA and quin-2. This result suggests that hydroxyperoxy-5,8,11,13-icosatetraenoic acid may activate a Ca2+-dependent nuclease. The addition of the antioxidant enzymes CuZn-superoxide dismutase and catalase had no effect, while glutathione peroxidase suppressed strand breakage by 90%. To our knowledge, our results yield a first insight into the mechanism of action of monocyte clastogenic factor and the role of inflammation in tumor promotion.