Increased ecto‐5′‐nucleotidase (ecto‐5′NT) protein expression in several multidrug‐resistant (MDR) cell lines, documented previously by our group, suggests that this enzyme is involved in drug resistance. Here, Northern blot analysis of selected cell lines and their MDR variants positively correlated ecto‐5′NT protein with its mRNA expression. An inhibitor of ecto‐5′NT enzymatic activity, α,β‐methyleneadenosine 5′‐diphosphate (AMP‐CP), was used to determine if functionally active enzyme had a role in drug resistance. AMP‐CP (0.3 mM) reversed the resistance of ecto‐5′NT‐positive MDR cells (MCF7/A6, L1210/A) to doxorubicin, whereas it did not affect the doxorubicin sensitivity of the ecto‐5′NT‐negative parental cell lines or that of 2 ecto‐5′NT‐negative MDR cell lines (HL60/VCR and A2780/DX5). Furthermore, AMP‐CP increased rhodamine uptake and inhibited rhodamine efflux from ecto‐5′NT‐positive MDR cells without affecting ecto‐5′NT‐negative MDR cells. The presence of exogenous adenosine (0.5 μM) circumvented AMP‐CP‐induced inhibition of rhodamine efflux from EL4/ADM cells. AMP‐CP inhibited the growth of the ecto‐5′NT‐positive L1210/A MDR cells but had no effect on the growth of the parental cell line. Determination of intracellular ATP levels indicated that MDR cells which had increased ecto‐5′NT expression also had a lower intracellular ATP level than their parental cells. Our results suggest that, in certain MDR cell lines, ecto‐5′NT serves as a required accessory molecule in resistance mediated by ATP‐dependent mechanisms and that growth‐sustaining nucleosides are provided by this salvage pathway. © 1996 Wiley‐Liss, Inc.