Nucleoside transport processes can be classified by the transport mechanism, e = equilibrative and c = concentrative, by the sensitivity to inhibition by nitrobenzylthioinosine (NBMPR), s = sensitive and i = insensitive, and also by permeant selectivity. To characterize nucleoside transport in acute promyelocytic NB4 cells, nucleoside transport was resolved into different components by selective elimination of transport processes with NBMPR and with Na'-deficient media. Initial transport rates were estimated from time course experiments. For adenosine, uridine, and formycin B, equilibrative transport accounted for approximately 60% of their uptake, with ei and es transport contributing almost equally, and Na+-dependent transport accounting for the remaining 40% of the total uptake. Thymidine uptake was mediated exclusively by equilibrative systems with ei and es systems each contributing 50% to total uptake. Adenosine accumulated above equilibrative concentrations, suggesting that a concentrative transport process was active and/or that metabolism led to adenosine's accumulation. Formycin B, a nonmetabolizable analog, also accumulated in the cells, supporting the concentrative potential of the Na+-dependent transporter. Kinetic analyses also provided evidence for three distinct high affinity transport mechanisms. NBMPR binding assays indicated the presence of two high affinity (K, 0.1 0 and 0.35 nM) binding sites. In conclusion, NB4 cells express ei and es transport, as well as a large ci transport component, which appears to correspond to cif (f = formycin B or purine selective) nucleoside transport, not previously described in human Cells. 8 1996 Wiley-Liss, Inc.