Two glycosphingolipids, GSL-1 and GSL-3, were isolated from Sphingomonas capsulata and studied by methylation analysis, laser desorption mass spectrometry, and 1 H and 13 C NMR spectroscopy, including two-dimensional 1 H, 1 H COSY and heteronuclear 13 C, 1 H COSY experiments. GSL-1 and GSL-3 differ in their carbohydrate part, their structures being a-d-GlcpA-(131)-Cer and a-d-Galp-(136)-a-d-GlcpN-(134)-a-d-GlcpA(131)Cer, respectively. Variations occur in the ceramide of GSL-1 and GSL-3, both having the same long-chain bases, erythro-2-amino-1,3-octadecanediol (sphinganine), (13Z)-erythro-2-amino-13-eicosene-1,3-diol and (13Z)-erythro-2-amino-13,14-methylene-1,3-eicosanediol, in the ratios 2.6 : 1 : 3.5 in GSL-1 and 1 : 1.2 : 1.5 in GSL-3. All bases are quantitatively substituted by amide-linked (S)-2-hydroxymyristic acid.