A new HPLC assay was developed to measure UDP-glucose dehydrogenase (UDP-GDH) activity in crude homogenates of 3T3 fibroblasts. UDP-GDH activity is directly related to the proliferative activity of the cell culture: enzyme activity is highest in log phase cells and decreases as the culture approaches quiescence. Serum stimulation of quiescent 3T3 fibroblasts results in an increase in UDP-GDH activity that has two components that are differentially affected by inhibitors of protein synthesis. Following serum stimulation, changes in cellular UDP-glucuronic acid concentrations mirror changes in UDP-GDH activity. UDP-xylose is a potent inhibitor of UDP-GDH but inhibitory concentrations of UDP-xylose could not be detected in cell extracts.