We developed three daunorubicin (D1)-resistant sublines (ML1/I, II, III) from the human myelocytic cell line (ML1). These sublines were 28-, 70- and 162-fold more resistant than sensitive (ML1/S) cells to the cytotoxicity of D1 and were cross-resistant to adriamycin, epiadriamycin, actinomycin D, VP-16, VM26, and mitoxantrone. Steady-state levels of D1 in resistant sublines I and II, in the presence or absence of azide, were not significantly different from those of sensitive cells. However, the steady-state level of D1 in subline III was significantly increased in the presence of sodium azide. D1 efflux was minimal in ML1/S and resistant cells in the absenced of glucose. Addition of glucose enhanced D1 efflux only in subline III. Verapamil increased the cellular levels of D1 and inhibited its efflux from resistant III cells but not from ML1/S cells. Verapamil also greatly enhanced the cytotoxicity of D1 for sublines I, II, and III. The differences between sensitive and resistant cells in D1 uptake and retention seemed inadequate to cause 162-fold resistance and suggested other factors may be contributing to the development of resistance. In support of this hypothesis, daunorubicin reductase activity was significantly lower in resistant cells than in ML1/S cells. The greatest decrease in activity occurred at pH 8.5 which represents aldehyde reductases. Currently, we are investigating other possibilities for D1 metabolism, such as aglycone and free radical formation.