The determination of six methylpyrazines was performed using high-performance liquid chromatography (HPLC). Methylpyrazines were simultaneously extracted and injected onto a silica gel column with a syringe-type minicolumn packed with diatomaceous earth granules. The extraction-injection solvent used was dichloromethane and the mobile phase solvent for HPLC was dichloromethane containing 0.08% of 1.65 M ammonia solution and 0.5% of methanol. Methylpyrazines were detected using an ultraviolet detector set at 275 nm. Linear relationships between the amount of sample and peak height were confirmed from 50 ng/ml to 10 micrograms/ml of the biofluids. When an aliquot of 10 microliters of biofluid was introduced to the minicolumn, the detection limit of methylpyrazines was as low as 30 ng/ml with each pyrazine derivative. The method is simple and accurate and is thus applicable to pharmacokinetic studies which are performed on animals. The results showed that the possible pharmacological effects of methylpyrazines might be evaluated pharmacokinetically using this newly developed technique.