A simple and sensitive method for determination of 4'-epimeric UDP-sugars using ion-pair reversed-phase HPLC has been developed. The method presents advantages over existing ion-exchange HPLC procedures mainly concerning sensitivity and rapidity of analysis as well as efficiency and stability of the column. It is based on the ability of borate ions to react with cis-diols resulting in the formation of UDP-sugar-borate complexes with different charges. Good resolution and rapid separation (5-25 min) of all 4'-epimeric UDP-sugars tested was achieved with this method, was suitable for concentrations over 20 pmol. The applicability to biochemical analysis was demonstrated by the quantitative determination of the UDP-2-deoxyglucose and UDP-2-deoxygalactose formed in yeast cells upon incubation in the presence of 2-deoxygalactose.