The ability of macrophages to induce drug-resistant mutants was studied in an in vitro, macrophage-tumor cell coculture system utilizing the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus as measured by resistance to 6-thioguanine. Tumor cells of the metastatic mouse mammary tumor line 66 were sensitive to macrophage induction of thioguanine resistance as shown by an increase in the frequency of thioguanine resistant variants which arose following macrophage coculture to levels at least 5 to 10 fold above the spontaneous frequency. This increased frequency was not seen in a series of related, generally nonmetastatic lines. Detection of increased numbers of variants depended upon the macrophage to tumor cell ratio, with 50:1 or greater being necessary. The activity of the macrophages was dependent upon their activation stage. The induction of drug-resistant variants could be inhibited by oxygen radical scavengers. The basis for the emergence of thioguanine resistant cells appeared to be induction of new variants rather than selection of pre-existing resistant cells from the parental population since thioguanine sensitive and resistant cells were equally sensitive to macrophage mediated toxicity. In 6 of the 6 macrophage-induced variants tested, resistance was associated with loss of HGPRT activity. The reverse mutation frequency rate at the HGPRT locus in 5 macrophage-induced variants was low and similar to that of a stable, ethyl methanesulfonate-induced thioguanine resistant line, suggesting that macrophage induction of thioguanine resistance was the result of a true mutation, rather than an epigenetic event. Macrophages isolated directly from growing mammary tumors, as well as activated peritoneal macrophages, were capable of inducing thioguanine resistance in line 66 cells.