Biotransformation of 2-methoxyethanol (2-ME) by alcohol and aldehyde dehydrogenases is an established factor in the toxicity of this useful solvent. Little is known about potential capacity for 2-ME biotransformation by testis or other target tissues. We detected appreciable capacity for 2-ME biotransformation by alcohol dehydrogenase in testes from Sprague-Dawley rats. However, kinetic analysis showed a 6-fold lower affinity for 2-ME by alcohol dehydrogenase of testis compared to liver. 2-ME biotransformation was also detected in testes from Wistar rats and one strain of mice but not in testes from hamsters, guinea pigs, rabbits, dogs, cats or humans. Testes from all these species readily converted the aldehyde metabolite of 2-ME to 2-methoxyacetic acid. Hepatic capacities for 2-ME biotransformation by alcohol dehydrogenase varied from 22 to 2.5 mumol/mg prot/min with a species rank order of: hamsters >> rats = mice > guinea pigs = rabbits. There was no consistent concordance between activities for 2-ME versus ethanol, the prototype substrate for alcohol dehydrogenase, which could reflect substrate preferences of different isozymes. Species differences between rats and hamsters were also found for testicular and hepatic biotransformation of the glycol ethers, 2-ethoxyethanol and 2-butoxyethanol. Although species differences in capacity for 2-ME biotransformation were found, the observations do not provide an explanation for reported species and strain differences in susceptibility to 2-ME toxicity.