For the four anthracyclines idarubicin, daunorubicin, epirubicin and doxorubicin the passive and active efflux rates in intact multidrug resistant cells were compared. Although highly similar structurally, these anti-tumor agents differ in lipophilicity and membrane permeability (k). The method we used was based on the continuous measurement of the cellular efflux and determination of the ratio (RVp) of transport rates just before and just after inhibition of the active transport with verapamil (Vp). Hence, RVp - 1 should reflect the active transport rate relative to the passive transport rate. If cells were single, well-stirred compartments, RVp - 1 should equal Vmax/(k.Km), where Vmax is the maximal pumping rate and Km is the Michaelis constant. However, using the plasma membrane permeabilizing agent digitonin, we found an effective intracellular anthracycline store. Particularly, when the efflux was fast, e.g. with idarubicin or in intensively pumping cells, the intracellular transport began to control the cellular efflux. Under these conditions, k underestimated the true plasma membrane permeability (k0) and RVp - 1 underestimated Vmax/(k.Km). Based on the effects of digitonin on the efflux rates in pumping and nonpumping cells, we developed an index (RVp,corrected - 1) which should equal Vmax/(k0. Km). The term Vmax/(k0.Km) varied substantially between the drugs. It appears that differences in lipophilicity between the drugs do not affect passive efflux and pumping equally. This demonstrates that passive permeation plays a substantial and independent role in determining the drug resistance for these anthracyclines. The methods developed here enable dissection of this role from that of drug pumping and intracellular subcompartmentation.