A high performance liquid chromatography (HPLC) method has been developed for the measurement of uridine diphosphate galactose (UDPGal) and uridine diphosphate glucose (UDPGlc) in erythrocytes and cultured skin fibroblasts of normal controls and galactosemia patients. The method incorporates an internal standard, UDPxylose, and alkaline phosphatase for the removal of nucleoside phosphates in the regions of UDPGal and UDPGlc in the chromatographic system. UDPGal and UDPGlc were separated on dual Dionex Carbo Pac PA-1 anion exchange columns. Samples derived from galactosemia patients without any detectable erythrocyte galactose-1-phosphate uridyl transferase (GALT) activity (GG), had in comparison to the controls significantly lower levels of UDPGal in both erythrocytes and cultured skin fibroblasts (P = 0.0001), while galactosemia patients with detectable GALT activity (GV) had normal levels of UDPGal. GG patients on oral uridine therapy had normal levels of UDPGal. These findings are consistent with our previous reports using an enzymatic method. In terms of absolute values, the values for both UDPGal and UDPGlc in erythrocytes by HPLC analysis were lower than those reported with the enzymatic method; however, in cultured skin fibroblasts, the values were similar with the two methods.