We investigated the pharmacokinetics of epirubicin, an anthracycline derivative antibiotics, after intravenous (i.v.) bolus administration in rats. To analyze epirubicin levels in the plasma, bile, urine and tissue samples, we developed an high-performance liqid chromatography (HPLC)-based method which was validated for a pharmacokinetic study by suitable criteria. The plasma concentration of epirubicin after i.v. bolus administration was rapidly disappeared within 10 min from the blood circulation. The mean plasma half-lives at ${alpha}$ phase ($t_{1/2{alpha}}$) when administered at the dose of 2, 5, 10, 25 and 50 mg/kg were 2.14-2.61 min. The values of $t_{1/2{eta}}$ at the corresponding doses increased two folds (from 150 to 291 min) with increasing doses. The $CL_t$ values significantly decreased with the increase in dose. In contrast, $V_{dss}$ values increased about 1.5 times with the increase in dose from 2 to 50 mg/kg. Of the various tissues, epirubicin mainly distributed to the kidney, lung, heart and liver after i.v. bolus administration. The epirubicin concentrations in various tissues at 24 h after i.v. bolus administration were below $1.0{mu}g/g$ tissue. Epirubicin was excreted largely in the bile after i.v. bolus administration at the dose of 2, 10 and 50 mg/kg. The cumulative amount of epirubicin in the urine 72 h after dosage represented 20 % of the amount excreted in the bile 12 h after high dosage, indicating that i.v. administered epirubicin was mainly excreted in the bile. In conclusion, epirubicin was rapidly cleared from the blood circulation and transferred to tissues such as the kidney and liver 2 h after i.v. bolus administration. Moreover, the majority of epirubicin appears to be excreted in the bile by 12 h after i.v. bolus administration.