The uptake of cytidine, of thymidine and of uridine in brush border vesicles prepared from the cortex of rat kidney has been studied by the technique of rapid filtration. The nucleosides were not metabolized in the vesicles. The time-courses of uptake in the presence of inwardly directed gradients of Na+ and of K+ showed an overshoot, indicating uphill transport. The overshoot was much more pronounced with Na+ than with K+; it was not observed when Na+ was at equilibrium across the membrane. The uptake of the nucleosides was stimulated by an inside negative potential in the presence of Na+. These results provide evidence for a cotransport of pyrimidine nucleosides with Na+. The apparent Km's for the uptake of cytidine, of thymidine and of uridine were 3.76 mumol X 1(-1), 4.18 mumol X 1(-1) and 7.21 mumol X 1(-1) respectively. The uptake of the pyrimidine nucleosides was insensitive to 6-nitrobenzylthioinosine. This insensitivity as well as the high affinity for the nucleosides and the capacity for uphill transport indicate that the nucleoside carrier(s) is renal brush border is (are) different from the carriers found in most other cell types.