The multidrug resistance gene (mdr1) is a member of the recently described ATP binding cassette (ABC) superfamily of transporters. Family members include: (1) the cystic fibrosis transmembrane conductance regulator gene; (2) the hlyB gene of bacteria, and (3) the histocompatibility antigen modifier (HAM) gene. The level of expression of mdr1 correlates with multidrug resistance (MDR), the ability of cells to efflux otherwise toxic doses of several chemotherapeutic agents. MDR activity is also associated with the efflux of cationic lipophilic compounds such as the fluorescent dye rhodamine 123. Recently it was reported that normal lymphocytes efflux rhodamine 123, suggesting that these cells possess MDR-like activity due to the expression of mdr1. In this study, using two-color flow cytometric analysis, we observed that the ability to efflux rhodamine 123 was heterogeneous among human lymphocyte subsets in the order of CD8 greater than CD4 greater than CD2O. Rhodamine 123 efflux and accumulation in lymphocytes was sensitive to the known MDR reversing agents, verapamil and Solutol HS 15. Collectively, these data suggest that an MDR-like transport system is present in normal lymphocytes and may be important for trafficking of molecules involved in lymphocyte function.