We examined the influence of incubation time or cytosolic protein concentration on the metabolite production of 5-fluorouracil (5FU). Although the activity of dihydropyrimidine dehydrogenase (DPDase) from rat liver is considered to be retained for up to 60 min, the production rate of 5FU metabolites was reduced, probably due to depletion of the substrate in 40 mumol/l and lower concentration of 5FU. Since the ratio of the metabolite production rate to the cytosolic protein became smaller in higher concentrations of cytosolic protein, the DPDase activity should be compared in the same concentration of cytosolic protein. The production rate of 5FU metabolites was considered to be linear up to 40 mumol/l 5FU incubated with 500 micrograms cytosolic protein. The rate of the metabolite production calculated by one-point sampling significantly correlated with the enzyme activity by the multi-point sampling method. Minimizing sampling points to determine the DPDase activity would save time and expense.