Doxorubicin (DOX) is an anthracycline antibiotic that possesses broad-spectrum antineoplastic activity, and is one of the most important anticancer agents. The purpose of this study was to investigate the effects of cyclosporine A (CsA) on the brain regional distribution of DOX and its liposome DOX formulation (Lipo-Dox). Liquid chromatography with tandem mass spectrometry (LC-MS/MS) was used to measure DOX in rat plasma and in various brain regions (cerebral cortex, hippocampus, striatum, midbrain, cerebellum, and the rest of brain). Good linearity was achieved over the 5-5000ng/mL range, with coefficients of correlation greater than 0.995. The limit of quantification for doxorubicin was 5ng/mL. This study was divided into the following four groups: DOX alone, DOX+CsA, Lipo-Dox alone and Lipo-Dox+CsA. After administering DOX (5mg/kg, i.v.) alone and DOX+CsA (10mg/kg, i.v.), it was undetectable in various brain regions. When the same dose of Lipo-Dox (5mg/kg, i.v.) and Lipo-Dox+CsA (10mg/kg, i.v.) were given individually, the plasma level and the brain regional level of DOX were much greater than those of DOX given alone. These results indicate that Lipo-Dox prolongs the DOX level in plasma and enhances brain distribution of DOX. The disposition of DOX might be regulated by P-glycoprotein.