The intestinal absorption and the site for hydrolysis of pivampicillin were compared with those of ampicillin by in situ ligated loop method in rats, using the radioactive compounds labeled at the phenylglycyl and the oxymethylene moieties. It was shown that pivampicillin is well absorbed from all parts of intestine, while ampicillin is absorbed from a more limited part of intestine. Pivampicillin was found to be rapidly transferred from the lumen into the intestinal wall as an intact ester and was hydrolyzed rapidly in the tissue. Ampicillin thus formed appeared to be transitorily accumulated in the tissue before being transferred into the portal venous blood gradually. Ampicillin was absorbed slowly without any accumulation in the intestinal wall. Microautoradiography of pivampicillin-¹⁴C in the intestine demonstrated that the radioactivity is accumulated in the epithelial cells in a high concentration. Histochemical and cytochemical detection of non-specific esterase revealed that a high activity is located specifically in the epithelial cells of villi and that the activity is localized in the membranes of endoplasmic reticulum as well as the cytoplasm, while there was almost no activity in the microvilli and terminal web region. It is considered therefore that pivampicillin is penetrated into the epithelial cells without any cleavage of the ester group and hydrolyzed by non-specific esterase in the apical cytoplasm of the cells. When the esterase activity was compared histochemically in rats, dogs and monkeys, it was found that the activity was extremely weak in dog intestine, providing an explanation for the fact that intact pivampicillin was detected in the portal venous blood only in dogs after oral administration. A possible relation between these results and the fact that a hepatic toxicity of pivampicillin was observed only in dogs among these animals was pointed out and discussed.