Hydrophobic column chromatography of bovine brain extracts (40-80% ammonium sulfate fraction) on immobilized colchicine resulted in the selective elution of one major protein with decreasing ionic strength of medium. This protein was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.1) on the basis of its biochemical properties, N-terminal amino-acid sequence and enzymatic activity. The present method enabled GAPDH to be isolated with a high recovery (80%; 184 mg/kg brain) and could be of potential use for the purification of GAPDH from various tissues.