Activated macrophages have been shown to inhibit the incorporation of 125IUDR into the DNA of Lewis lung (LL) tumor cells and to lead to the cytotoxicity of these target cells. Despite this apparent inhibition of DNA synthesis, the increase in the number of LL cells cultured with activated macrophages paralleled that of LL cells alone for the first 18 hours of culture (approximately one doubling of cells). To resolve this paradox, the DNA content of LL cells, which were cultured with activated macrophages for 16 hours, was analyzed by flow microfluorometry. These experiments indicated that subsequent to co-culture with activated macrophages, LL cells with a 50% reduction in their normal DNA content could be detected. Furthermore, after a 2-hour incubation with colcemid, a greater than 95% reduction in metaphase cells occurred in LL cells cultured with activated macrophages. However, dividing nuclei without chromosome condensation appeared to be a prominent feature of these cultures. This would suggest that the genotypic or phenotypic program for cell division in the tumor cells is such that under these unique circumstances of tumor cell-activated macrophage interaction, cell division can proceed in the absence of DNA synthesis. We suggest that this aberrant division may be related to the lethal event which leads to activated macrophage-mediated tumor cell cytotoxicity.