A simplified protocol to obtain fatty acid methyl esters (FAME) directly from fresh tissue, oils, or feedstuffs, without prior organic solvent extraction, is presented. With this protocol, FAME synthesis is conducted in the presence of up to 33% water. Wet tissues, or other samples, are permeabilized and hydrolyzed for 1.5 h at 55 degrees C in 1 N KOH in MeOH containing C13:0 as the internal standard. The KOH is neutralized, and the FFA are methylated by H(2)SO(4) catalysis for 1.5 h at 55 degrees C. Hexane is then added to the reaction tube, which is vortex-mixed and centrifuged. The hexane is pipetted into a gas chromatography vial for subsequent gas chromatography. All reactions are conducted in a single screw-cap Pyrex tube for convenience. The method meets many criteria for fatty acid analysis, including not isomerizing CLA or introducing fatty acid artifacts. It is applicable to fresh, frozen, or lyophilized tissue samples, in addition to oils, waxes, and feedstuffs. The method saves time and effort and is economical when compared with other methods. Its unique performance, including easy sample preparation, is achieved because water is included rather than eliminated in the FAME reaction mixtures.