The emergence of drug-resistant tumor cells is a major limiting factor in cancer chemotherapy. There is little information about the nature of such resistant variants among human cancer cell populations. Doxorubicin (DOX)-resistant sublines of the human sarcoma cell line MES-SA were selected by continuous in vitro exposure to DOX. Stepwise increases in DOX concentration produced variants which were 25- and 100-fold resistant to DOX. These sublines displayed marked cross-resistance to daunorubicin, dactinomycin, mitoxantrone, colchicine, vincristine, vinblastine, and etoposide and moderate resistance to mitomycin C and melphalan. Cross-resistance was not observed, however, to methotrexate, 5-fluorouracil, bleomycin, carmustine, or cisplatin. DOX resistance in these cell lines appeared to be stable despite long periods of growth in drug-free medium. Two additional marker chromosomes were identified in the 100-fold resistant variant, which indicated clonal selection during drug exposure, but no double minute chromosomes or homogeneously staining regions were noted. Doxorubicin accumulation in the DOX-resistant cells was reduced by approximately 50% compared to that of the sensitive MES-SA cells, as a result of enhanced efflux of DOX from the resistant cells. There was no evidence of appreciable DOX metabolism by either the sensitive or resistant cells. These studies demonstrate marked DOX resistance and multidrug resistance arising in a human sarcoma line during exposure to DOX. The pleiotropic nature of this resistance is similar to that described in other models. Decreased drug accumulation due to enhanced drug efflux is identified as a major mechanism of resistance in these cells, although other factors may also be involved.