A multidrug-resistant variant of the human HL-60 promyelocytic leukemia cell line (HL-60/MX2) has been isolated in vitro by subculturing these cells in progressively increasing concentrations of mitoxantrone. The MX2 cells are cross-resistant to etoposide, teniposide, bisantrene, dactinomycin, 4'-(9-acridinylamino)methanesulfon-m-anisidide, and the anthracyclines daunorubicin and doxorubicin but retain sensitivity to the Vinca alkaloids melphalan and mitomycin C. In addition, the MX2 cells display slight collateral sensitivity to bleomycin. Despite being 30-35-fold less sensitive to mitoxantrone, net [14C]mitoxantrone accumulation at 60 min was reduced by only 10% in the mitoxantrone-resistant cells compared to the parental line. Furthermore, at later time points, e.g., 120 and 180 min, mitoxantrone accumulation in the MX2 cells exceeded that in HL-60 cells by 8.5 and 6.4%, respectively. No significant differences were observed between the sensitive and resistant cell lines in the initial (first 60 s) accumulation of mitoxantrone, and only minor (3-6%) enhancement of mitoxantrone efflux was detected in the resistant cell type. Monoclonal antibodies to P-glycoprotein had no detectable reactivity with membrane vesicles from either the sensitive or resistant cell types as determined by standard immunoblotting techniques. The mitoxantrone-resistant cells displayed a reciprocal translocation [rcpt(1;3)-(q21;p23)] not found in the sensitive parent, but there were no demonstrable double minute chromosomes or homogeneous staining regions in cells from either line. Thus, these mitoxantrone-resistant human leukemia cells display many features which are atypical for the "classic" multidrug resistance phenotype and should provide a useful model for the study of multidrug resistance which is not mediated by P-glycoprotein.