We have shown previously that macrophages are mutagenic to bacteria (A. M. Fulton et al., Cancer Res., 44: 4308-4311, 1984) and can induce the appearance of drug-resistant variants of murine mammary tumor cells (K. Yamashina et al., Cancer Res., 46: 2396-2401, 1986). The present study asks whether inflammatory macrophages can induce lesions in the DNA of cocultured tumor cells and seeks to determine the mediators of this damage. We quantitated the induction of DNA strand breaks using the technique of fluorometric analysis of DNA unwinding. We report that inflammatory macrophages coincubated with a mammary tumor cell line for 60 min at a 1:1 ratio result in significant numbers of strand breaks in the tumor cell DNA. The degree of damage is equivalent to 300 to 1200 rads of gamma-irradiation. Resident (unstimulated) peritoneal macrophages also induce tumor cell DNA strand breaks. However, inhibitor studies reveal quantitative and qualitative differences in strand breaks induced by inflammatory (elicited) versus resident peritoneal macrophages. Resident macrophages require a longer induction period (60 min) before significant breaks are detected, but induce more breaks than do elicited macrophages, which require only a 5-min coincubation period to induce significant damage. The enzyme catalase, which removes H2O2, protects tumor cells from both macrophage effector populations as does the prostaglandin synthase inhibitor, indomethacin. The superoxide anion scavenger, superoxide dismutase, and the lipoxygenase inhibitor, nordihydroguaiaretic acid, are protective against resident macrophage effects only. The metal chelator, o-phenanthroline, provides limited protection for elicited macrophages but induces total DNA breakage in the presence of resident macrophages. Taken together, our data indicate that the degree of strand breakage is greater for the macrophage population with high arachidonate metabolism and low oxidative metabolism (resident macrophages) and less for the macrophage population with high oxidative and low arachidonate metabolism (MVE-2 elicited macrophages). Inhibitor studies implicate both metabolites of reactive oxygen and arachidonate as mediators of this tumor cell DNA damage, with the relevant mediator dependent upon the particular macrophage population under study.