Fish oil triglycerides were transesterified to their corresponding methyl and ethyl esters. The esters were separated by reversed-phase high-performance liquid chromatography on a lo-cm column of 5-pm octadecylsilyl silica, using a ternary mobile phase of acetonitrile-tetrahydrofuran-water (9:5: 11). The separation of principal saturated and unsaturated C&C& fatty acid esters was accomplished in 60 min. For the polyenoic acid esters, detection limits of cu. 26 pg were observed, and baseline resolution of the important critical pair of esters of the 03 fatty acids, eicosapentaenoic acid (C20503) and docosahexaenoic acid (C22:603) was achieved. However, to obtain baseline resolution of the esters from minor fatty acid esters, a urea crystallization procedure was required prior to chromatographic analysis. The influence on the reversed-phase chromatographic retention of four different ester moieties on the fatty acids was also studied. The n-alkyl esters exhibited an increase in In k' with increasing chain length and with increasing lipophilic character of the alkyl ester moieties; however, with the aromatic benzyl derivatives there was a decrease in In k' compared with the alkyl esters.