Metabolism of lenampicillin hydrochloride (LAPC), especially ampicillin (ABPC) structure of LAPC, was investigated after oral administration in human, dogs and rats. The unchanged compound was not detected in blood and urine, furthermore in animal portal vein after oral administration of LAPC in human and 2 animals. Therefore, LAPC seemed to be rapidly hydrolyzed during the process of absorption. The intestinal absorption of LAPC was satisfactory in view of the urinary excretion of metabolites, accounting for 93% of dose in human, 74% in dogs and 55% in rats, respectively. It could be judged by the bioautograms and the correlation between bioassay and HPLC determination of ABPC that the active metabolite in blood or urine was only ABPC. The major urinary metabolites were ABPC, alpha-aminobenzylpenicilloic acid (ABPA) and 5S-penicilloic acid isomer (5S-ABPA) in human and 2 animals, but the differences were observed on the excretion ratio between human, dogs and rats. LAPC was stable in the intestinal contents, but liable to hydrolyze in the intestinal wall, blood and liver of rats. From the facts described above, it was concluded that LAPC was the efficient prodrug of ABPC in terms of the enhancement of absorption and decrease of side effects.