Valspodar is a P-glycoprotein (P-gp) inhibitor widely used in preclinical and clinical studies for overcoming multidrug resistance (MDR). In rat, Valspodar showed properties of low hepatic extraction and wide distribution, similar to that of its structural analogue cyclosporine A. Administration of Valspodar to animals before mitoxantrone treatment increased the accumulation of mitoxantrone in the MDR tumors to 94% of that in the wild-type tumors. These studies have added direct in vitro and in vivo visual information on how P-gp processes anticancer compounds and how P-gp inhibitors modulate MDR in resistant cancer cells.