Salvage of circulating nucleosides provides an alternative to de novo synthesis of nucleotides and may modify response to antimetabolites. We have investigated treatment with uridine phosphorylase as a means of inhibiting salvage of uridine in vivo. Examination of the metabolism of intravenous [3H] uridine in mice revealed that 30-40% was salvaged by conversion to uracil nucleotides and the remainder was catabolized. In contrast, less than 0.3% of intravenous [3H]uracil was salvaged. Addition of partially purified bacterial uridine phosphorylase to plasma produced a rapid phosphorolysis of uridine. In vivo, 1.5 hr after intravenous injection of 9 units of uridine phosphorylase, plasma activity (1.3 units/ml) was 65-fold greater than that of control mice. Pretreatment with uridine phosphorylase prior to administration of [3H]uridine produced a marked (65-92%) but incomplete inhibition of salvage of uridine in all tissues examined. The dose required to produce 50% inhibition of uridine salvage at 1 hr was 2 to 2.5 units/mouse. The inhibition of nucleoside salvage by this approach may permit an evaluation of the role of nucleoside salvage in the supply of cellular nucleotides and the effects of concurrent inhibition of de novo and salvage pathways for nucleotide synthesis.