An improved two column method for rapid manual determination of glutamate (Glu), glutamine (Gln), gamma-aminobutyric acid (GABA) and aspartate (Asp) from brain regions is presented. The method uses a Dowex-1 column and various concentrations of acetic acid to separate Glu, Asp and the neutral amino acids. The neutral amino acids are subsequently placed on a Dowex-50 column from which potassium acetate buffers are used to separate the amido amino acids, GABA and the other neutral amino acids. The amido amino acids are hydrolysed in NaOH and the glutamine derived glutamate is separated from contaminants on another Dowex-1 column. Amino acids are manually quantitated using the fluorescence from the reaction with o-phthaldialdehyde. The method described in this paper requires no special equipment other than a fluorometer. The procedure was designed to determine levels of amino acid neurotransmitters and catecholamines from a single brain sample as small as 0.12 g, however, due to the sensitivity of the o-phthaldialdehyde reaction, levels of the four amino acids could be determined on a sample one tenth that size or smaller.