Overexpression of multidrug resistance-associated protein (MRP) has been detected in resistant cell lines derived from a variety of tumor types. The deduced amino acid sequence of MRP suggests that it is a member of the ATP-binding cassette transmembrane transporter superfamily that may be glycosylated and/or phosphorylated [S. P. C. Cole et al., Science Washington, DC), 258: 1650-1654, 1992]. Recently, transfection of HeLa cells with MRP expression vectors has demonstrated that the protein is capable of increasing resistance to natural product drugs such as anthracyclines, Vinca alkaloids, and epipodophyllotoxins (C. E. Grant et al., Cancer Res., 54: 357-361, 1994). Although the resistance phenotype of the transfectants is similar to that of the human small cell lung cancer cell line, H69AR, from which MRP was originally cloned, the transfectants differ in their drug accumulation characteristics, relative resistance to certain drugs, and MRP mRNA:protein ratio. Such differences have also been observed among drug-selected cell lines that overexpress MRP, and the underlying causes of these variable phenotypes are presently not known. We have utilized polyclonal anti-MRP-peptide antibodies to compare MRP post-translational modification, stability, processing, and subcellular distribution in the HeLa transfectants and in the drug-selected H69AR cells. These studies establish that MRP in both the transfected and selected cells is an ATP-binding, integral membrane glycophosphoprotein with an apparent molecular weight of 190,000. No obvious differences were detected in the extent or type of glycosylation or the kinetics of processing and turnover of the protein that might contribute to the different characteristics of the transfected and drug-selected cells. Analyses of the subcellular distribution of MRP by isopyknic density gradient centrifugation revealed that approximately 80% of MRP in the HeLa transfectants was associated with a low density plasma membrane fraction while the comparable fraction in the drug-selected H69AR cells contained only approximately 50% of the protein. The remaining MRP and plasma membrane markers were codistributed in higher density fractions consistent with the presence of MRP in endocytotic vesicles. The relatively high proportion of MRP associated with these fractions in H69AR cells may contribute to the lack of an observable accumulation defect in these cells when compared with the transfectants.