A glc method utilizing a flame ionization detector was developed for determing secobarbital (I) in human plasma and urine. Extraction of plasma and urine to which the internal standard, allylcyclopental barbituric acid (II) had been added was accomplished using benzene. An on-column methylation technique was used to facilitate glc analysis. Accuracy and precision of the method was demonstrated over the concentration range anticipated in acute dosing human studies. The method was evaluated in one subject who received two different oral doses of I four weeks apart and found to be applicable to plasma and urine analysis. Saliva was also collected from this subject and analyzed by the method. Although I could be detected at one time interval, quantification could not be accomplished due to lack of sufficient sensitivity.