The uridine phosphorylase inhibitors, 5-benzylacyclouridine (BAU) and 5-benzyloxybenzylacyclouridine (BBAU) (Biochem. Pharmacol., 31: 1857, 1982), inhibited uptake of uridine in L5178Y cells. By a rapid sampling technique, BAU and BBAU were shown to inhibit the transport (zero-frans influx) of uridine, thymidine, and adenosine in human erythrocytes as well as in murine L5178Y cells. In all cases, competitive inhibitions were observed. values for the transport of adenosine, uridine, and thymidine in erythrocytes were 2.2, 195, and 199 MM, while Vm« were 2.9, 118, and 96.5 pmol/min/106 cells, respectively. In L5178Y cells, K values of 14.8 and 23.1 and V™ of 389 and 176 pmol/min/106 cells were obtained for adenosine and uridine, respectively. For erythrocytes, the K values of BAU were 127, 124, and 198 HM using adenosine, uridine, and thymidine as the substrate; and those of BBAU, 14.1 and 19.2 HM for adenosine and uridine, respectively. In L5178Y, the Kj values of BAU were 202 and 234 M, and those of BBAU, 39.8 and 27.9 fiM for adenosine and uridine, respectively. These data indicate that, in two cell types, K, values for BAU and BBAU did not vary regardless of the substrate used; that the values of Kj are different for the erythrocytes and L5178Y cells; and that BBAU is at least 5-fold more potent than BAU as an inhibitor of nucleoside transport. The inhibitory effects on the efflux of preloaded uridine indicate that BAU and BBAU are inhibitors, rather than permeants, of the nucleoside transport system. © 1984, American Association for Cancer Research. All rights reserved.