Abstract: AimA bioassay method was established for the determination of active concentrations of lidamycin and studied its pharmacokinetics in mice and dogs. MethodsCytotoxicity of lidamycin in vitro was used to determine drug serum concentrations in vivo. ResultsValidity of methodology met the requirements of pharmacokinetic study. The concentration-time profile in mice after iv lidamycin of 100, 50 and 10 μg·kg-1 was best fitted with 2-compartmental model with t_1/2α and t_1/2β of 0.77-1.8 min and 5.6-7.2 min, respectively. The AUC were 2 851.3, 887.8 and 166.4 μg·min·L^-1, respectively and increased with dose nonlinearly. There were similar trends between AUC and the potency of tumor growth inhibition. After iv lidamycin of 12 μg·kg^-1 in dogs, the concentrations of lidamycin decreased rapidly and the AUC was 16 μg·min·L^-1, which were lower and quicker than those in mice. The levels in serum after second administration at day 15, were lower than those of the first. ConclusionActive concentrations and pharmacokinetics of lidamycin were obtained by bioassay method successfully. There are species differences and single and multi-dosing differences in the pharmacokinetics of lidamycin.