Isolated stage VI oocytes from Xenopus laevis expressed uridine transport activity after microinjection of mRNA from rat jejunum. Uridine uptake during 30 min (10 microM, 20 degrees C) by mRNA-injected oocytes reached 2.5 pmol/oocyte, compared with endogenous uptake by water-injected oocytes of about 0.05 pmol/oocyte. The expressed transport activity was 96% Na(+)-dependent, saturable (apparent Km = 15 microM) and inhibited by phloridzin (IC50 = 100 microM). Nucleoside inhibition studies resolved the expressed transport activity into two components: 1) a novel Na(+)-dependent system of broad purine and pyrimidine specificity that was inhibited by low concentrations of guanosine, inosine, adenosine, uridine, thymidine, and cytidine and 2) a Na(+)-dependent system of narrower specificity that was inhibited by low concentrations of guanosine, inosine, adenosine, and uridine and by high concentrations of thymidine and cytidine. The characteristics of the latter system are consistent with those of the Na(+)-dependent nucleoside transport system N1 (cif), previously identified in a number of cell types and tissues, including intestinal epithelia and cultured cells of intestinal origin. The broad specificity system, which was also detected in mRNA-injected oocytes using thymidine as permeant, has been given the provisional designation N3 to distinguish it from the previously described N1 (purine-selective) and N2 (pyrimidine-selective) Na(+)-linked nucleoside transporters. Rat jejunal transporters N1 and N3 were both expressed maximally by the same mRNA size fraction (1.6-3.0 kb, peak 2.3 kb).