The transport of [U-14C]uridine was investigated in rat cerebral-cortical synaptosomes using an inhibitor-stop filtration method. Under these conditions the rapid efflux of uridine from the synaptosomes is prevented and uridine is not significantly metabolized in the synaptosome during the first 1 min of uptake. The dose-response curve for the inhibition of uridine transport by nitrobenzylthioinosine (NBMPR) was biphasic: approx. 40% of the transport activity was inhibited with an IC50 (concentration causing half-maximal inhibition) value of 0.5 nM, but the remaining activity was insensitive to concentrations as high as 1 microM. Similar biphasic dose-response curves were observed for dilazep inhibition, but both transport components were equally sensitive to dipyridamole inhibition. Uridine influx by both components was saturable (Km 300 +/- 51 and 214 +/- 23 microM, and Vmax. 12 +/- 3 and 16 +/- 3 pmol/s per mg of protein, for NBMPR-sensitive and NBMPR-insensitive components respectively), and inhibited by other nucleosides such as 2-chloroadenosine, adenosine, inosine, thymidine and guanosine with similar IC50 values for the two components. Inhibition of uridine transport by NBMPR was associated with high-affinity binding of NBMPR to the synaptosome membrane (Kd 58 +/- 15 pM). Binding of NBMPR to these sites was competitively blocked by uridine and adenosine and inhibited by dilazep and dipyridamole, with Ki values similar to those measured for inhibiting NBMPR-sensitive uridine influx. These results demonstrate that there are two components of nucleoside transport in our rat synaptosomal preparation that differ in their sensitivity to inhibition by NBMPR. Thus conclusions regarding nucleoside transport in rat brain based only on NBMPR-binding activity must be viewed with caution.