A gas chromatographic-negative ion chemical ionization mass spectrometric (GC-NCI-MS) method for the determination of flumazenil in plasma is described. The GC of flumazenil (M(r) 303) is considered to be difficult as it is readily adsorbed in the GC column. Therefore, preconditioning the GC column with reconstituted extract from plasma and Silyl-8 was required to cover the active sites on the column. Monitoring the maximum mass peak (m/z 275) of the flumazenil resulted in a tenfold enhancement of sensitivity and signal-to-noise ratio (concentration = 1 ng/ml). Isotopically labeled flumazenil-d3 (M(r) 306, m/z 278) was used as the internal standard. The detection limit for flumazenil was found to be 0.1 ng/ml with an injection volume of 2 microliters. The signal-to-noise ratio was about 10. The routine quantification limit was set at 2 ng/ml for dog plasma and 1 ng/ml for human plasma. The sample volumes in both instances were 1 ml.