The in vivo interaction of methylene chloride and its metabolites with F344 rat and B6C3F1 mouse lung and liver DNA was measured after inhalation exposure to 4000 ppm [14C]methylene chloride for 3 hr. DNA was isolated from the tissues 6, 12, and 24 hr after the start of exposure and analyzed for total radioactivity and the distribution of radioactivity within enzymatically hydrolyzed DNA samples. Covalent binding to hepatic protein was also measured. A further group of rats and mice were dosed intravenously with [14C]formate after exposure to nonradiolabeled methylene chloride for 3 hr to determine the pattern of labeling resulting from incorporation of formate into DNA via the C-1 pool. Low levels of radioactivity were found in DNA from lungs and livers of both rats and mice exposed to [14C]methylene chloride. Two- to fourfold higher levels were found in mouse DNA and protein than in rat. Chromatographic analysis of the DNA nucleosides showed the radioactivity to be associated with the normal constituents of DNA. No peaks of radioactivity were found that did not coincide with peaks of radioactivity present in hydrolyzed DNA from formate-treated rats and mice. Under the conditions of this study there was no evidence for alkylation of DNA by methylene chloride in either rats or mice.