The bioavailability of propranolol (PL) after oral administration of ester-type prodrug was compared in rat and dog, and the possible reason for species difference was investigated. In dog, the oral bioavailability of PL was enhanced by the use of prodrug due to saturation of metabolism of PL. In contrast, high (10 mg/kg) and low (2.5 mg/kg) doses of butyryl PL and isovaleryl PL failed to improve oral bioavailability of PL in rats. The hydrolase activities for prodrugs in rat liver were lower than those of dog (by 4-12-fold), but those of rat intestinal mucosa were significantly higher than those of dog (50-260-fold). Although it is clear from the in vitro hydrolysis using subcellular fractions that the rapid hydrolysis in intestinal mucosa was mainly due to cytosolic components, the brush-border membrane vesicle in rat intestine also showed hydrolase activity for both prodrugs. In situ absorption experiment in rat revealed an improvement in the apparent absorption rate of PL as the result of prodrug use (1.3-fold) and the nearly complete hydrolysis of isovaleryl PL during intestinal absorption, which is a slower hydrolyzed prodrug than butyryl PL in intestinal mucosa and liver. The defects for enhancing oral bioavailability in rats appears to be based on an unsaturation of metabolism for PL, which is derived from a decrease in PL concentration in hepatocytes, owing to rapid hydrolysis of the prodrug in intestinal mucosa and slow hydrolysis of the prodrug in liver. Furthermore, human intestinal mucosa showed a surprisingly high hydrolase activity in microsomes. Therefore, the oral bioavailability of PL after administration of prodrugs might be not significantly improved in human.