The hydrolysis of UDP-N-acetylglucosamine by extracts of rat embryo cells has been compared to the hydrolysis of this sugar nucleotide by extracts of hamster embryo cells. A method utilizing high-pressure liquid chromatography was developed to separate and quantify the products of the hydrolysis. Rat embryo cells as well as hamster embryo cells hydrolyzed UDP-N-acetylglucosamine virtually completely under the experimental conditions. The principal product of the hydrolysis of UDP-N-acetylglucosamine by hamster embryo cells was N-acetylglucosamine. The sugar moiety of N-acetyglucosamine, produced by hamster embryo cells, was identified by thin-layer chromatography to be glucose. By contrast, the major metabolite (60--70%) of the hydrolysis of UDP-N-acetylglucosamine by rat embryo cells was identified as a phosphorylated acetylamino sugar. Only about 30--40% of the substrate was degraded to the free acetylamino sugar. Examination of the sugar moiety of the phosphorylated and of the free acetylamino sugar by thin-layer chromatography indicated that it was a mixture of glucose and of the epimers, mannose or galactose. The identity of the epimers has as yet not been established. Thus, unlike hamster embryo cells, rat embryo cells contain an active epimerase. However, the phosphohydrolase of the rat embryo cell which degrades the phosphorylated acetylamino sugars to the free acetylamino sugars seems to be less active than the enzyme in the hamster embryo cell. On the basis of the data of this study a comparison of the pattern of hydrolysis of UDP-N-acetylglucosamine by normal and transformed embryonic cells of the rat and of the hamster appears feasible.