Liver microsomes from male Syrian golden hamsters and Sprague Dawley rats metabolize the cis and trans isomers of N-nitroso-2,6-dimethylmorpholine (NNDM) to N-nitroso-(2-hydroxypropyl)(2-oxopropyl)amine (HPOP) as the major product detectable by h.p.l.c. The rates of total metabolism are similar for both the cis and trans isomers; but the cis isomer of NNDM yields greater than 70% of the total product as HPOP while the trans isomer yields HPOP only as a minor product (20-30%) in both hamster and rat. The inability to identify other products could be attributed to alpha-hydroxylation which leads to fragmentation of NNDM and loss of tritium label to water. In order to investigate the possibility of the participation of an alpha-hydroxylation reaction, the metabolism of NNDM fully deuterated at either the 3 and 5 (alpha-d4) or the 2 and 6 (beta-d2) positions was examined and compared to the metabolism of the undeuterated compound (d0). Although the rates of metabolism of all the cis and trans derivatives of NNDM were similar (VMax = 2.13 nmol/min/mg hamster microsomal protein) as determined from measurements of substrate disappearance, the yields of HPOP were different. Maximum HPOP yields were observed with cis alpha-(d4) NNDM (93.9% of the total), followed by cis d0 NNDM (72.3%), trans alpha-(d4) NNDM (60.1%), trans d0 NNDM (30.2%), cis beta-(d2) NNDM (19.5%) and trans beta-(d2) NNDM (8.5%). These results suggest that alpha-hydroxylation is an alternative to beta-hydroxylation. Since the carcinogenic potency of the various deuterium derivatives of NNDM for the Syrian golden hamster parallels their ability to yield HPOP, beta-hydroxylation is closely related to pancreatic carcinogenesis in the hamster. Rat liver microsomal fractions showed the same patterns of HPOP formation to total metabolite yields as hamster liver microsomes with both the cis and trans isomers. However, rates of NNDM metabolism and HPOP formation were 7 times faster with hamster than with rat liver microsomes. Such a difference may be related to the failure of the cis isomer to induce pancreatic cancer in rats.