Chromatographic analysis of the acid soluble nucleotide pool in 3T3 fibroblasts 30 minutes after serum stimulation revealed one component that was increased compared to quiescent controls. This component was identified as UDP-glucuronide by chromatographic and chemical means. The time course and magnitude of the serum stimulated increase in UDP-glucuronide is similar to the time course and magnitude of the increase in [14C]uridine uptake. Other factors capable of stimulating [14C]uridine uptake, including epidermal growth factor, platelet derived growth factor, interleukin-1, and a phorbol ester also caused an increase in UDP-glucuronide. The results show that one of the earliest changes in pyrimidine nucleotide metabolism after mitogen stimulation is an increase in UDP-glucuronide synthesis, which may be related to increased uridine uptake.