To compare the activity of pyrimidine nucleoside phosphorylase (PNP), an enzyme involved in the metabolism of 5-fluorouracil (5-FU), we used uridine (Urd), deoxyuridine (dUrd), and thymidine (dThd) as substrates and human, rat, and mouse neoplastic and normal tissues. As PNP activity was higher in the tumor tissues than in the normal ones in all species examined, the level of PNP activity is expected to be one critical factor linked to the effectiveness of 5-FU. In rats and mice, the ratio of the activities of Urd, dUrd, and dThd was about 10:7:1, whereas in humans, the ratio was 1:30:20. The main enzyme of PNP is Urd phosphorylase in rodents and dThd phosphorylase in humans. Therefore, when examining the metabolism of 5-FU and its analogues for potential clinical application, human tissues should be used.