A simple, rapid and accurate method to extract and clean thiopental in biological materials rich in fat was developed by using a separation column packed with Extrelut and Florisil. A Quantitative determination of thiopental by means of a gas chromatograph equipped with a flame photometric sulfur detector was attempted. The calibration curve was linear over the range of 5-200 micrograms/ml. Extraction of replicate tissues involving 3 micrograms of thiopental resulted in a recovery of the 99.7-101.8% range and a coefficient of deviation of the 0.3-1.9% range. Replicate extraction of five tissues from rats treated with thiopental resulted in a coefficient of deviation of below 6%. In rats given 40 mg/kg of thiopental and sacrificed after 5 min, the thiopental levels in fat were found to be higher after a slow intravenous injection than after a quick injection. The same tendency, however, was not observed in other tissues. It seems that the rate of intravenous thiopental injections might be estimated by comparison with thiopental levels in fat tissues.