It has long been recognized that β-glucuronidase can hydrolyze the glycosidic linkage of a wide variety of O-glucuronides in vitro but no direct evidence has been available concerning their in vivo hydrolysis. The present investigation was undertaken to confirm whether or not the glucuronide, when administered intravenously, could be hydrolyzed by β-glucuronidase in vivo. For this purpose, p-nitrophenyl glucuronide was injected intravenously into a rat, and 24-hour urine was analyzed. This result showed that most of the administered dose was excreted unchanged, but a small portion was excreted as p-nitrophenol by the action of 4-glucuronidase in the bladder. By the effect of CCl₄ on the β-glucuronidase activity in vivo and in vitro, an evidence was obtained that such a difficult hydrolysis of the glucuronide in vivo was perhaps attributable to its difficult penetration into the cell.