We have developed a one-dimensional thin-layer chromatography procedure that resolves the initial substrate uracil and its catabolic derivatives dihydrouracil, N-carbamoyl-beta-alanine (NCBA) and beta-alanine. This separation scheme also simplifies the preparation of the radioisotopes of N-carbamoyl-beta-alanine and dihydrouracil. Combined, these methods make it possible to assay easily and unambiguously, jointly or individually, all three enzyme activities of uracil catabolism: dihydropyrimidine dehydrogenase, dihydropyrimidinase, and N-carbamoyl-beta-alanine amidohydrolase. Earlier reports had presented data suggesting that these three enzyme activities were combined in a complex because they appeared to be controlled at a single genetic locus [Dagg, C. P., Coleman, D.L., & Fraser, G.M. (1964) Genetics 49, 979-989] and because they appeared able to channel metabolites [Barrett, H.W., Munavalli, S.N., & Newmark, P. (1964) Biochim. Biophys. Acta 91, 199-204]. Although the three enzymes from rat liver have similar sizes, with apparent molecular weights of 218 000 for dihydropyrimidine dehydrogenase, 226 000 for dihydropyrimidinase, and 234 000 for NC beta A amidohydrolase, they are easily separated from each other. Kinetic studies show no evidence of substrate channeling and therefore do not support a model for an enzyme complex. The earlier reports may be explained by our studies on the amidohydrolase, which suggest that under certain conditions this enzyme may become the rate-limiting step in uracil catabolism.