Transport of radiolabeled mitoxantrone, a new antineoplastic agent, was studied using cultured mouse L1210 leukemia cells. The initial velocity of influx remained linear for about 90 sec and was 110 pmoles/10(6) cells measured at 60 sec. The steady-state accumulation of about 480 pmoles/10(6) cells was not reached until 30 min. The unidirectional drug influx was linear from 0 to 1000 microM extracellular drug concentration. The initial uptake was relatively temperature independent between 37 degrees and 27 degrees, but accumulation at steady state was 17% lower at 27 degrees. None of six metabolic inhibitors had an appreciable effect on initial uptake. Efflux was initially exponential with a half-life of 2.8 min; this efflux and the residual drug concentration plateau were not affected by KCN or verapamil. Under steady-state conditions, about 86% of the cell-associated label was contained in parent drug and the remainder in an unidentified metabolite. These studies indicate that the mechanism of mitoxantrone uptake is passive diffusion. The efflux is not energy requiring, but there is considerable tight binding of the drug to cellular structures.