The effect of several experimental conditions on methane (CH4) production from dimethylsulfoxide (DMSO) in incubation mixtures containing liver microsomes and NADPH generating systems was studied. The process was heat sensitive in part but a significant fraction was non-enzymatic in nature. CH4 formation from DMSO was not significantly modified by 2-diethylaminoethyl-2,2-diphenylvalerate. HC 1 (SKF 525A) or EDTA 1 mM and significantly enhanced under an atmosphere of (CO 80% + O2 20%) rather than under air. A marked increase in CH4 production was observed when paraquat (PQ) was included in incubation mixtures but not when nifurtimox (Nfx) was added. Results support the hypothesis of hydroxyl free radical (.OH) formation during PQ biotransformation but cast doubts about its production for the case of Nfx. The low temperature gas chromatographic separation of d3-CH4 from CH4 described opens the future possibility for detecting trace formation of .OH in vivo, without interference from fecal CH4 formation by administering d6-DMSO to animals and collecting exhaled gases produced, in chambers containing the entire animal.