Brimonidine is an alpha 2-adrenergic agonist that is efficacious in lowering intraocular pressure in humans. A highly sensitive and selective gas chromatography/mass spectrometry (GC/MS) assay is described for quantitation of brimonidine in human plasma following ocular installation. Brimonidine in 1 ml of plasma was extracted together with tetradeuterated brimonidine (internal standard) and clonidine (carrier) by solvent extraction. After solvent evaporation, 3,5-bis(trifluoromethyl)benzoyl derivatives were formed and injected onto a GC/MS apparatus under negative chemical ionization conditions. The ions monitored for derivatized brimonidine and tetradeuterated brimonidine were m/z 691 [M-HBr] and m/z 694 [M-DBr], respectively. Calibration curves were linear from 2 to 1000 pg ml-1 (r2 = 0.981-0.996). The method was specific for brimonidine relative to endogenous compounds in plasma. The inter-day relative standard deviation for analysis of quality controls was 12% or less, and the inter-day assay accuracy ranged from 97 to 104% of nominals. The GC/MS assay showed adequate sensitivity for analysis of human samples from volunteers ocularly dosed with 0.5% brimonidine tartrate solution. Overall, the GC/MS assay showed excellent precision and accuracy, and a minimum quantifiable concentration of 2 pg ml-1.