N,N-dimethylacetoacetamide (DMAAm) is a β-dicarbonyl compound used as an industrial intermediate. This study investigated the disposition and metabolism of [¹⁴C]DMAAm in male rats and female mice. A single oral dose of [¹⁴C]DMAAm (target dose of 10 or 130 mg/kg) was administered to male F344 and Wistar-Han rats. [¹⁴C]DMAAm was almost completely absorbed and excreted in urine, with ca. 80-90% of the dose recovered within 24 h for both rat strains. Fecal excretion and CO₂ exhalation were minimal (1 and 2%, respectively). Less than 3% of the dose remained in tissues at 24 h. There was no apparent dose- or strain-related difference in the disposition of [¹⁴C]DMAAm in rats. In female B6C3F1 mice administered 8 mg/kg [¹⁴C]DMAAm, 80% of the administered radioactivity was recovered in urine and cage rinse in 24 h. Urinary metabolites were isolated and characterized by liquid chromatography /mass spectrometry following oral administration of 435 mg/kg [(¹⁴C]DMAAm in male F344 rats. Metabolism occurred via reduction of the 3-keto group and oxidation of the N-methyl groups, to give N,N-dimethyl-3-hydroxybutanamide, N-methyl-N-hydroxymethyl-3-hydroxybutanamide, and N-hydroxymethyl-3-hydroxybutanamide, and N-demethylation to give N-monomethylacetoacetamide (MMAAm).