A convenient, specific, rapid method, developed for the determination of ochratoxin in barley, involves initial extraction of the sample with water-chloroform (1+9), followed by chromatography of the extract on an aqueous sodium bicarbonate-diatomaceous earth column. The ochratoxin esters are removed from the column with hexane-chloroform. Elution of the column with formic acid-chloroform (1+99) yields ochratoxins A and B. The ochratoxin esters are purified further on an aqueous sodium bicarbonate-methanol-diatomaceous earth column. The extracts are then quantitated on TLC plates by measuring fluorescence intensity. The presence of ochratoxins A and B is confirmed through formation of the corresponding ethyl ester derivatives. In an intralaboratory study, in which samples spiked at 25, 50, and 100 μg/kg levels were analyzed by 4 analysts, the average recovery for ochratoxin A was 81.2% with a lower detection limit of 12 μg/kg.