Gliotoxin, a mycotoxin with antimicrobial and immunosuppressive capabilities, is produced by several genera of fungi including the pathogenic fungus Aspergillus fumigatus. The ability of selected isolates of A. fumigatus to produce gliotoxin on three different media was tested and a thin layer chromatographic and high performance liquid chromatographic method for quantitation of gliotoxin from rice culture was developed and is described. Rice cultures were extracted with chloroform and the resulting extract was partially purified by precipitation with petroleum ether and cleanup by gel permeation chromatography. Gliotoxin was detected by thin layer chromatography and quantitated by high performance liquid chromatography using a U.V. absorbance detector with a 254 nm filter and a mobile phase of methanol-water 43:57 (V/V) with a flow rate of 2.0 ml/min. The retention time for gliotoxin was approximately 4.8 min. From rice samples spiked with gliotoxin concentrations of 0.67, 1.33, 2.67, 4.00 and 5.33 micrograms/g the average recovery was 83.8%.