Results of previous studies in this laboratory and others have suggested that L-carnitine is degraded in the gastrointestinal tract of the rat, perhaps by the action of indigenous flora. L-[methyl-14C]Carnitine was administered to rats either orally or intravenously in doses of 86 nmol or 124 mumol, and expired air, 48-h urine and fecal collections, and selected tissues at 48 h after isotope administration were examined for radiolabeled carnitine and metabolites. Urine and feces of rats receiving oral L-[methyl-14C]carnitine consistently contained two radiolabeled metabolites which were identified as trimethylamine N-oxide (primarily in urine) and gamma-butyrobetaine (primarily in feces). In these rats, these metabolites accounted for up to 23% and 31% of the administered dose, respectively. By contrast, for rats receiving intravenous L-[methyl-14C]carnitine or germ-free rats receiving the isotope orally or intravenously, virtually all of the radioactivity recovered was in the form of carnitine. Analyses for 14CO2 and [14C]trimethylamine in expired air revealed little or no (less than 0.1% of dose) conversion to these compounds, regardless of size of dose or route of administration. Results of this study demonstrate conclusively that L-carnitine is degraded in the gastrointestinal tract of the rat and that indigenous flora are responsible for these transformations.