The in vivo metabolism of the cis and trans isomers of N-[3,5-3H]nitroso-2,6-dimethylmorpholine (NDMM) was studied in female Fischer rats, Syrian golden hamsters and guinea pigs by analysis of urinary metabolites using high pressure liquid chromatography (HPLC). Animals were treated by gavage with 12 mg/kg body wt. of NDMM, composed of both isomers and 12 microCi/kg body wt. of either of the separated radioactive isomers (cis or trans). Control animals received 12 mg, 12 microCi/kg body wt. NDMM with both isomers labeled in their natural proportion. There was a substantial increase in the excretion of a particular metabolite, 2-(2-hydroxyl-methyl)ethoxy propanoic acid, in the urine of rats, hamsters and guinea pigs 24 h after received the trans isomer (24, 22 and 13% of the total dose excreted, respectively). A minor metabolite was determined to be 2,6-dimethylmorpholine-3-one, another product of alpha-oxidation. The metabolite 1-amino-2-hydroxypropanol was identified, indicating that NDMM was metabolized by both alpha- and beta-oxidation. In all three species, animals administered the cis isomer excreted larger amounts of N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine (HPOP) and N-nitroso-bis(2-hydroxypropyl)amine (BHP) products of beta oxidation, than those treated with the trans isomer. Hamsters and guinea pigs treated with the more carcinogenic cis isomer in these species, also excreted twice as much of two other metabolites than was found in the urine of animals given the trans isomer. The trans isomer of NDMM appeared to be preferentially metabolized by alpha-oxidation and from earlier studies this metabolic pathway seemed to be important in carcinogenesis by NDMM in the rat. The cis isomer might be in a conformation more favorable for beta-oxidation and this pathway may be of primary importance in carcinogenesis by NDMM in hamsters and guinea pigs.