To elucidate the interaction between hydrophilic lithospermic acid B and lipophilic tanshinone II A in rats. A reliable high-performance liquid chromatography method was adopted for simultaneous determination of lithospermic acid B and tanshinone II A in rat plasma, through which the pharmacokinetic interaction between lithospermic acid B and tanshinone II A by intravenous injection was investigated. The simultaneous intravenous injection of tanshinone II A and lithospermic acid B significantly altered the pharmacokinetic parameters of both compounds when compared with the individual intravenous administration of each compound. The area under the concentration-time curve of tanshinone II A and lithospermic acid B increased by 18.35 and 59.31%, respectively. The mean retention time of tanshinone II A and lithospermic acid B increased, respectively, from 9.3 to 32.8 h and 20.2 to 49.1 h. The concomitant use of tanshinone II A magnified the volume of distribution at steady state (V ss ) and time for the drug in the plasma to reduce the highest concentration by half (t 1/2 ) of lithospermic acid B, while at the same time the V ss and t 1/2 of tanshinone II A changed significantly in the presence of lithospermic acid B. Lithospermic acid B and tanshinone II A interact with each other following simultaneous intravenous injection in rats and this observation may expand the clinical use of Danshen (Radix Salviae Miltiorrhizae).