Inhalation of ¹⁴C-labeled methyl chloride (CH₃Cl) by rats resulted in accumulation of radioactivity in lipid, RNA, DNA, and protein isolated from lung, liver, kidney, testes, brain, muscle, and intestine. At the end of a 6-hr exposure to 500 or 1500 ppm ¹⁴CH₃Cl, 8 to 20% of the radioactivity present in whole tissue homogenates was associated with acid-insoluble material. Most of this activity represented labeling of protein and lipid, although the concentration of ¹⁴C was usually over 10-fold higher in nucleic acids, per mole of nucleotide, compared with protein, per amino acid residue. Sephasorb HP chromatography of DNA revealed that radioactivity derived from ¹⁴CH₃Cl was associated with normal purine bases and no methylated bases were detected. Similarly, 82% of the radioactivity present in the lipid fraction copurified with the major species of phospholipids upon thin-layer chromatography. Pretreatment of rats with cycloheximide reduced the amount of CH₃Cl-derived radioactivity associated with tissue protein by 42 to 58%, whereas [³H]leucine incorporation was inhibited by 75 to 85%, indicating that most, but not necessarily all, of the uptake of ¹⁴CH₃Cl into protein was dependent on normal protein synthesis. Pretreatment with methotrexate inhibited the uptake of ¹⁴CH₃Cl into lipid, acid-insoluble material, RNA, and DNA by 47, 64, 65, and 93%, respectively. Pretreatment with methanol inhibited ¹⁴CH₃Cl uptake into acid-insoluble material by 66%. These findings indicate that most of the ¹⁴C appearing in macromolecules following inhalation of ¹⁴CH₃Cl arose through metabolic incorporation, stemming from metabolism of CH₃Cl via the one-carbon pool. Methanol apparently competed with CH₃Cl for entry into this pool, although the failure of ethanol, 4-methylpyrazole, or 3-amino-1,2,4-triazole to inhibit ¹⁴CH₃Cl incorporation indicated that CH₃Cl was not metabolized to methanol per se. Methanol pretreatment also inhibited ¹⁴CO₂ evolution from ¹⁴CH₃Cl, indicating that metabolism of CH₃Cl via single-carbon pathways may be of major quantitative significance.