The activities of five enzymes, orotate phosphoribosyltransferase (OPRTase), uridine kinase (UR kinase), thymidine kinase (TdR kinase), uridine phosphorylase (UR Prylase) and thymidine phosphorylase (TdR Prylase), were examined in subcultured human acute leukemia cell lines (HL-60, CCRF-CEM), subcultured human solid tumor cell lines (Colo-205, HeLa-S3) and human cancerous tissues with a view to compare the activation of 5-fluorouracil in them. There was no significant difference in the activity of any enzyme between HL-60 and CCRF-CEM, Colo-205 and HeLa-S3, and human lung cancerous tissue and human colon cancerous tissue. Compared between the acute leukemia cell lines and the solid tumor cell lines, the UR kinase activity was high in both cell lines. The OPRTase and UR Prylase activities were low in the solid tumor cell lines. In the cancerous tissues, both the UR kinase and TdR kinase activities were low, but the UR Prylase and TdR Prylase activities were markedly high. The results suggest that the intracellular activation of 5-fluorouracil varies with different human cancerous cells. When the anti-cancer activity of 5-fluorouracil is tested in vitro, the difference of fluoropyrimidine metabolism in subcultured cell lines from that in the cancerous tissue should be taken in account.