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A precise and reproducible high-performance liquid chromatographic method for the determination of pentylenetetrazol in serum and brain tissue is described. The procedure employs reversed-phase chromatography, monitoring the eluant at 202 nm. Quantification is based on peak-height ratio of the drug to the internal standard (p-methylphenobarbital). A linear response is obtained to 100 micrograms/ml (serum) or micrograms/g (brain tissue). Within-day and between-day precision are smaller than 5%, and analytical recovery is greater than 95%. Numerous drugs tested do not interfere with the assay. The method has been used to investigate the kinetics of pentylenetetrazol distribution in serum and in discrete areas of rat brain.