Equilibrative nucleoside transport processes in mammalian cells are categorized as either nitrobenzylthioinosine (NBMPR)-sensitive (es) or NBMPR-insensitive (ei). Inhibition of the es process arises from binding of NBMPR to a high-affinity site(s) on the es transporter that can be identified by photoaffinity labeling with [3H]NBMPR. This study examined the equilibrative nucleoside transport processes of cultured human erythroleukemia (K562) cells. The presence of NBMPR binding sites (4.8 +/- 0.9 x 10(5)/cell, Kd = 0.3 nM), together with the identification of polypeptides by specific photolabeling of membranes with [3H]NBMPR, indicated that K562 cells possess es nucleoside transporters (ca 500,000 copies/cell). The photolabeled polypeptides of K562 cells migrated with lower relative mobility (peak M(r) value, 63,000) than did those of human erythrocytes (peak M(r) value, 53,000). This difference in apparent M(r) was abolished by prolonged treatment of membrane proteins with N-glycosidase F, suggesting that equilibrative nucleoside transport in K562 cells and erythrocytes is mediated by the same, or a closely related, es isoform. A cDNA encoding the es nucleoside transporter of human placenta (termed hENT1) was recently isolated by a strategy based on the N-terminal sequence of the es transporter of human erythrocytes. hENT-like mRNA species were detected in K562 cells, as well as in several other human cell lines of neoplastic origin (A459, G361, HeLa, HL-60, Molt-4, Raji, SW480), by high-stringency northern analysis with a placental hENT1 probe. A cDNA that encoded a protein identical to hENT1 was isolated by reverse transcriptase polymerase chain reaction with primers specific for hENT1. NBMPR inhibited zero-trans influx of 3H-labeled adenosine, uridine and thymidine by 50% (IC50 values) at 0.4-1.0 nM, confirming the presence of an NBMPR-sensitive (es) transport process, which accounted for 80-90% of total transport activity. The remaining component was identified as the equilibrative NBMPR-insensitive (ei) transport process since it: (i) exhibited low (IC50 > 1.0 microM) sensitivity to NBMPR; (ii) was not concentrative; and (iii) was unchanged by elimination of the sodium gradient. The kinetic parameters (determined at 37 degrees C) for the es- and ei-mediated processes differed markedly. Values for transport of uridine by the es- and ei-mediated processes were, respectively: K(m) = 229 +/- 39 and 1077 +/- 220 microM; Vmax, 186 +/- 31 and 40 +/- 5 pmol/microliter cell water/sec. Values for transport of adenosine by the es and ei-mediated processes were, respectively, 61 +/- 9 and 133 +/- 17 microM; Vmax, 70 +/- 5 and 23 +/- 8 pmol/microlitere cell water/sec. The ei-mediated process, although small, was of pharmacologic importance since K562 cells could not be protected by NBMPR (10 microM) from the cytotoxic effects of tubercidin (7-deazaadenosine).