A new procedure was developed to measure uridine and cytidine in plasma. These nucleosides are present in micromolar concentrations in the plasma of rats, mice and humans. Inhibitors of pyrimidine synthesis de novo (pyrazofurin or N-phosphonacetyl-L-aspartate) produce only modest decreases in the concentration of circulating uridine or cytidine in the rat. Since uridine and cytidine are rapidly cleared from the circulation of the rat, constant infusions of radiolabeled uridine and cytidine were used to establish a steady-state specific activity of circulating nucleoside without altering the normal endogenous concentration. These studies permitted an estimation of the contribution of circulating pyrimidine nucleoside to the nucleotide pools of various rat tissues. Most of the uridine entering the circulation (> 70%) is catabolized rather than salvaged by formation of nucleotides. Cytidine in the circulation is much more efficiently utilized and is predominantly salvaged. The implication of these results for chemotherapy based on inhibition of pyrimidine synthesis de novo is discussed.