To establish an HPLC method for determination of catalpol in CSF (cerebrospinal fluid) of rats.Rats were intravenously injected 1.0 g x L(-1) catalpol physiological saline, and the sample of CSF from subarachnoid space of the cerebrum 40 minutes of injection. The sample of CSF from normal rats was used for blank control, the all samples were preserved in a refrigerator of - 20 degrees C, and use HPLC was employed to determine the catalpol content. The separation of catalpol was performed on Hypersil C18 reversion phase chromatographic column. The mobile phase consisted of water-acetonitrile (99.5: 0.5) with a flow rate of 1.0 mL x min(-1) and detection wavelength of 210 nm.The linear range of catalpol in CSF was 0.5-40 mg x L(-1) (r = 0.999 7). The absolute recoveries were (90.2 +/- 1.71)%, (89.1 +/- 1.17)% and (86.9 +/- 0.98)%; and the methodological recoveries were (99.8 +/- 1.98)%, (101.1 +/- 3.04)%, (100.1 +/- 2.30)% respectively. The within-day and between-day derivation RSD were less than 4%. Catalpol was stable in a refrigerator of -20 degrees C for 15 days.The method is simple and accurate for the determination of the content of catalpol in CSF.