The metabolism of 3-hydroxy-3-methylindolenine (HMI), a recently discovered metabolite of 3-methylindole (3MI, skatole) produced by porcine liver microsomes, was investigated in vitro using porcine liver cytosol. HMI was rapidly metabolized to a single product, 3-hydroxy-3-methyloxindole (HMOI), by porcine cytosol. By the use of the selective inhibitors menadione and quinacrine, it was shown that the enzyme responsible for the oxidation of HMI into HMOI was aldehyde oxidase (AO; aldehyde:oxygen oxidoreductase, EC 220.127.116.11). The activity of AO in the conversion of HMI to HMOI was measured in a population of pigs (n = 30) with a wide range of 3MI levels in back fat (0.07-0.30 mg/kg). AO activity was found to be negatively correlated (r = -0.70; P < 0.001) with the level of 3MI in fat. The results of the present study suggest that AO plays an important role in the metabolism of 3MI in the pig and that its catalytic activity is related to an adequate 3MI clearance.