Effect of vitamin B2 deficiency on rat liver dihydropyrimidine dehydrogenase was investigated. It was found that the purified enzyme contains 2 mol flavin per molecule, which consists of equal proportions of flavin-adenine dinucleotide (FAD) and riboflavin 5'-phosphate (FMN). When rats were fed on a vitamin B2-deficient diet for 5 weeks, dihydropyrimidine dehydrogenase activity in the liver was diminished, followed by a decrease in enzyme concentration. Moreover, the addition of exogenous FAD or FMN did not restore the activity. Thus endogenous flavin may regulate the enzyme half-life or synthesis. Lowering of dihydropyrimidine dehydrogenase activity in the livers of rats fed on a vitamin B2-deficient diet did not affect the uridine, uracil and sigma UMP (the sum of acid soluble uracil 5'-nucleotides) pool in liver.