We report structure determination of an octaene fatty acid, 4,7,10, 13,16,19,22,25-octacosaoctaenoic acid (28:8n-3). The molecular weight and double bond locations were determined using acetonitrile chemical ionization mass spectrometry (MS) and MS/MS and were confirmed by MS of hydrogenated and deuterogenated 28:8 and by argentation thin-layer chromatography. 28:8n-3 was 1.2 +/- 0.1%, in oil derived from the heterotrophic dinoflagellate Crypthecodinium cohnii and a commercial polyunsaturated fatty acid concentrate derived from fish oils (0.16 +/- 0.01%), both components of human dietary supplements. It was not found in whole bovine retina, cultured Y79 human retinoblastoma cells, or neonate baboon cerebral cortex. The long chain polyunsaturates present in the C. cohnii oil suggest a possible route for 28:8n-3 biosynthesis similar to that for biosynthesis of 22:6n-3.