A GLC-mass spectometric method employing specific-ion monitoring was developed for the determination of plasma concentrations of labeled (15N1,3, 13C2) and unlabeled barbital following simultaneous intravenous and oral administration. This method proved to be more sensitive and precise than the method employing GLC with flame-ionization detection or GLC with alkali flame-ionization detection. After extraction of [15N1,3, 13C2]barbital, barbital, and the internal standard, butalbital, from plasma with ether, the organic solvent is evaporated, and the labeled and unlabeled drug as well as the internal standard are converted into their N,N-dimethyl derivatives by treatment with diazomethane. The excess reagent is evaporated, and the resulting methyl derivatives are analyzed by GLC-mass spectrometry with selected-ion monitoring. The method is sufficiently sensitive to determine 0.5 microgram of the labeled and unlabeled drug/ml with a relative standard deviation of less than 5%. The application of the method to the determination of the plasma concentration of labeled and unlabeled drug over 6 days following simultaneous oral and intravenous administration of a single dose is demonstrated.