We recently reported that glycolipid compositions of gadoid fish nerve tissues are unique in the abundance of 1) galactosyl diacylglycerol and its sulfate ester; 2) glucosyl ceramide; and 3) fatty acid ester of cerebroside (J. Lipid Res. 1992. 33: 1351-1359). The present paper reports the characterization of less polar glycolipids isolated from Alaskan pollack brain. Of twelve glycolipids purified by column chromatography, four were of the galactosyl diacylglycerol type. Chemical analysis, infrared spectrometry, and a permethylation study followed by gas chromatography-mass spectrometry revealed that they were 1,2-di-O-acyl-3-beta (6-O-acyl-D-galactopyranosyl)-sn-glycerol and 1,2-di-O-acyl-3-beta(2'-O-acyl-D-galactopyranosyl)-sn-glycerol, and mixtures of these two isomers, with slightly different fatty acid compositions, respectively. The other eight less polar glycolipids were pure forms or mixtures of isomeric forms of cerebroside fatty acid esters in which the substituted position of the acyl group on the hexose moiety varied. The permethylation study revealed that they were 6-O-acyl-beta-D-galactopyranosyl, 6-O-acyl-beta-D-glucopyranosyl, 2-O-acyl-galactosyl, 2-O-acyl-glucosyl, 3- or 4-O-acyl-galactosyl, and 3-O-acyl-glucosyl ceramides, the 6-O- and 2-O-acyl isomers being predominant. This is the first report of the natural occurrence of 1) acyl galactosyl diacylglycerol in animal tissues and 2) acyl glucosyl ceramide in nerve tissues.