Nucleoside transport was determined in the cloned porcine kidney cell line PK-15 which exhibits properties of tubular cells. The cells did not express any Na(+)-dependent, concentrative nucleoside transport. They exhibited only nitrobenzylthioinosine-sensitive equilibrative nucleoside transport. Their transport activity, however, was only about 10% of that observed in many other mammalian cell lines. This low transport activity correlated with a relatively low number of high-affinity nitrobenzylthioinosine binding sites (5 x 10(3)/cell). Furthermore, although the equilibrative transporter of PK-15 cells exhibited a similar broad substrate specificity as the equilibrative nucleoside transporters of other mammalian cells, it exhibited a much higher affinity for certain nucleosides, especially cytidine and uridine, than the latter. The Michaelis-Menten constants for zero-trans transport and equilibrium exchange of uridine in ATP-depleted cells were about the same (about 40 microM), indicating equal mobility of the nucleoside-loaded and empty carrier. Concentrative nucleoside transport was detected in one set of PK-15 cultures, but was found to be due to mycoplasma contamination.