Acetanilide with the three hydrogens of the acetyl group replaced by deuterium was administered to 9 human volunteers (50 mg p.o.) and 5 male Sprague-Dawley rats (10 mg kg-1 p.o. and 100 mg kg-1 p.o.) and urine samples collected for 8 and 48 h, respectively. Capillary gas chromatography/mass spectrometry (GC/MS) was used to determine if the major metabolite was deuterated paracetamol or a mixture of this compound with paracetamol produced by deacetylation followed by reacetylation. Acetyl group exchange of the parent compound was also studied. Conjugates of the metabolite were hydrolysed and the free compound derivatized to an O-propionyl ester before GC/MS analysis. Paracetamol containing no deuterated acetyl group was detected in all studies, together with the labelled metabolite. The mean ratio of deuterated to unlabelled paracetamol in man was approximately 8:1 and was independent of acetylator status. The acetyl group exchange measured in the rat was time-dependent but was always significantly higher than in man. The mean ratios of deuterated to unlabelled paracetamol in urine samples collected 0-4 h and 24-48 h after administration of the higher trideuteroacetanilide dose to the rats were approximately 2:1 and 4:1, respectively.