Aflatoxin B1 (AFB1) must be activated to the electrophilic AFB1 exo-8,9-epoxide to be genotoxic and carcinogenic. A role for epoxide hydrolase in detoxication has been suggested but never directly addressed. In light of recent studies determining the instability of AFB1 exo-8,9-epoxide in H2O, a role for epoxide hydrolase appears dubious. Rat liver or recombinant rat epoxide hydrolase provided an enhancement to the already fast hydrolysis rate of up to 22%. Purified human epoxide hydrolase provided no detectable enhancement to the rate of chemical hydrolysis. Some reduction in the genotoxicity of AFB1 was observed when the ratio of rat epoxide hydrolase to cytochrome P450 was high (approximately 50-fold). An 80-fold excess of human epoxide hydrolase over cytochrome P450 only produced an effect of approximately 25% inhibition. It appears, therefore, that there is little evidence to support a role for epoxide hydrolase in the detoxication of AFB1.