Rat brain, obtained 10 min after death, contained high levels of endogenous γ-aminobutyric acid (GABA) and glutamic acid. Incubation of this brain homogenate at 37°C indicated decrease of GABA with time due to degradation by GABA-transaminase. Reported high-performance liquid chromatographic (HPLC) methods for glutamic acid decarboxylase (GAD) assay depend on the difference between the GABA content of the reaction mixture after and before the incubation period. None of the methods considered the degradation of GABA during incubation. Furthermore, during determination of the Michaelis constant (KM) for the reaction none of them considered the endogenous substrate. Here we have focused on these factors which seriously affect the maximum velocity (Vmax) and KM values during GAD assay by the HPLC technique. By a simple and rapid HPLC technique we have measured GAD activity in post-mortem rat brain after removing endogenous glutamic acid by charcoal treatment and using gabaquline to prevent GABA degradation during incubation period. By this method a Vmax value of 46±4 nmol/h/mg protein and a KM value of 7.5±0.6 mM were observed for GAD activity of crude brain homogenate. For a comparative study, we have carried out radiometric assay of GAD activity from the same sample and observed a Vmax of 48±6 nmol/h/mg protein and KM of 6.9±0.4 mM. © 1991.