Acrylamide (AA) is a widely studied industrial chemical that is neurotoxic, mutagenic to somatic and germ cells, and carcinogenic in rodents. The recent discovery of AA at ppm levels in a wide variety of commonly consumed foods has energized research efforts worldwide to define toxic mechanisms, particularly toxicokinetics and bioavailability. This study compares the toxicokinetics of AA and its epoxide metabolite glycidamide (GA) in serum and tissues of male and female B6C3F1 mice following acute dosing by intravenous, gavage, and dietary routes at 0.1 mg/kg AA or intravenous and gavage dosing with an equimolar amount of GA. AA was rapidly absorbed from oral dosing, was widely distributed to tissues, was efficiently converted to GA, and increased levels of GA-DNA adducts were observed in liver after complete elimination from serum. GA dosing also resulted in rapid absorption, wide distribution to tissues, and produced liver DNA adduct levels that were approximately 40% higher than those from an equimolar dose of AA. While oral administration was found to attenuate AA bioavailability to 23% from the diet and to 32-52% from aqueous gavage, a first-pass effect or other kinetic change resulted in higher relative internal exposure to GA when compared to the intravenous route. A similar effect on relative GA exposure was also evident as the administered dose was reduced, which suggests that as dosing rate decreases, the conversion of AA to GA is more efficient. These findings are critical to the assessment of genotoxicity of AA at low doses in the food supply, which appears to depend on total exposure to GA.