L1210 mouse leukemia cells exhibit two distinct types of nucleoside transport activity that have similar kinetic properties and substrate specificity, but differ markedly in their sensitivity to the inhibitor nitrobenzylthioinosine (NBMPR) (Belt, J. A. (1983) Mol. Pharmacol. 24, 479-484). It is not known whether these two transport activities are mediated by a single protein or by separate and distinct nucleoside transport proteins. We have isolated a mutant from the L1210 cell line that has lost the NBMPR-insensitive component of nucleoside transport, but retains NBMPR-sensitive transport. In the parental cell line 20-40% of the nucleoside transport activity is insensitive to 1 microM NBMPR. In the mutant, however, uridine and thymidine transport are almost completely inhibited by NBMPR. Consistent with the loss of NBMPR-insensitive transport, the mutant cells can be protected from the toxic effects of several nucleoside analogs by NBMPR. In contrast, the toxicity of the same analogs in the wild type cells is not significantly affected by NBMPR, presumably due to uptake of the nucleosides via the NBMPR-insensitive transporter. On the other hand, NBMPR-sensitive transport in the mutant appears to be unaltered. The mutant is not resistant to cytotoxic nucleosides in the absence of NBMPR and the cells retain the wild type complement of high affinity binding sites for NBMPR. Furthermore, the affinity of the binding site for the inhibitor is similar to that of parental L1210 cells. These results suggest that NBMPR-sensitive and NBMPR-insensitive nucleoside transport in L1210 cells are mediated by genetically distinct proteins. To our knowledge this is the first report of a mutant deficient in NBMPR-insensitive nucleoside transport.