Murine myeloma SP2/0-Ag14 cells possess both nitrobenzylthioinosine (NBMPR)-sensitive and NBMPR-insensitive equilibrative uridine transport systems. No Na + -dependent uridine transport system was detected. The NBMPR-insensitive transport system is similarly insensitive to inhibition by dilazep and dipyridamole. Dose-response curve for the inhibition of equilibrative uridine transport by N-ethylmaleimide (NEM), a sulfhydryl reagent, in these cells was biphasic. About 30-40% of the uridine transport was inhibited by NEM at IC 5 0 value of 0.15 mM. The other 60-70% of the transport activity remained insensitive to NEM at concentration as high as 3 mM. The decrease in NBMPR-sensitive uridine transport in the presence of 0.3 mM NEM was due to a 3-fold decrease in transport affinity. Apparent K m values of 500 and 1600 μM and V m a x values of 13 and 12 μM/s were obtained for untreated and NEM-treated cells, respectively. NEM (0.3 mM) has little effect on the K m of NBMPR-insensitive transporter, with apparentK m values of 100 and 110 μM and V m a x values of 3.0 and 2.5 μM/s for untreated and NEM-treated cells, respectively. High sensitivity of NBMPR-sensitive transporter to NEM inhibition was also observed in HL-60 and MCF-7 cells. Decrease in specific 3 H-NBMPR equilibrium binding affinity in myeloma cells was observed after treatment with 0.3 mM NEM. Apparent K d values of 0.32 and 2.3 nM withB m a x values of 48 000 and 44 000 sites/cell were obtained for untreated and NEM-treated cells, respectively. NBMPR, dilazep and dipyridamole at 30 μM, and uridine at 10 mM failed to protect the NBMPR-sensitive transporter against NEM inhibition. It is possible that a critical sulfhydryl residue is closed to substrate binding/transporting site of the NBMPR-sensitive transporter. NEM, a sulfhydryl reagent containing an activated double bond, hinders the affinity of this transporter by forming a stable thiol ether bond with the reactive residue.