A reversed-phase high performance liquid chromatographic method for the determination of metronidazole (MTZ) in rabbit serum and cerebro-spinal fluid has been developed. The method is simple and convenient. The analysis was carried out with Zorbax C8 column, NaH2PO4(0.0075mol/L, pH 4.0)-CH3OH(4:11) as mobile phase and UV detector at 320nm, by using hydrochlorothiazide as internal standard. The standard curve is linear over the range from 0.5ng/L to 80.0ng/L in sample with correlation coefficient of 0.9998. The recovery of metronidazole from rabbits serum and cerebrospinal fluid are 97.1% +/- 1.5% and 97.4% +/- 1.6% respectively. The minimum detectable quantity is 80ng/mL in sample (S/N = 3). Under the chromotographic condition, the consistency of MTZ of the serum and cerebrospinal fluids of rabbits were detected separately after taking MTZ (0.1mg/g) orally. The drug-time curve was obtained and a satisfactory result was achieved. The new findings provide conditions for further exploration of the process of pharmacokinetics.