We describe here a simple, sensitive, selective and reproducible assay method for quantitative determination of aspartate, glutamate, serine, glutamine, glycine and gamma-aminobutyric acid in rat brain using reversed-phase high-performance liquid chromatography. The method is based upon formation of phenylthiocarbamyl derivatives of the amino acids. Good resolution of the six amino acids and the internal standard norvaline is achieved within 40 min. Other amino acids which have been reported to be present in rat brain do not interfere with the analysis. Standard curves for each of the amino acids exhibited good linearity (r greater than 0.9993) over the range 0.5-20 nmol. The coefficient of variation for the intra-day and inter-day determinations ranged from 0.4% at the highest to 11% at the lowest concentration limit. Storage of whole brains at -0 degrees C for up to 8 weeks did not affect mean concentrations of the six amino acids.