The insertion mode of the long fatty acid chain of the asymmetric glycosphingolipid C26:0-cerebroside sulfate (C26-CBS) in symmetric matrices of phosphatidylcholines of different acyl chain length has been investigated by transmission and attenuated total reflectance (ATR) infrared spectroscopy. The concentration of C26-CBS in myelin is increased in the demyelinating disease adrenoleukodystrophy. The conformational order and the orientation of the chains of the asymmetric glycosphingolipid have been evaluated for C26-CBS incorporated at 8 mol % in perdeuterated dimyristoylphosphatidylcholine (DMPC-d54) and perdeuterated dipalmitoylphosphatidylcholine (DPPC-d62). The results, for the gel phase, are consistent with interdigitation of the C26-CBS long acyl chain across the bilayer center of an all-trans-DMPC bilayer in which DMPC is less tilted than in the absence of CBS. In contrast, in DPPC the results suggest that although the CBS long chain interdigitates across the center of the bilayer, it does not change the tilt angle of the DPPC molecules in the gel phase. Furthermore, in DPPC, C26-CBS is less well oriented than the host DPPC molecules and it increases the gauche content of the DPPC acyl chains. The observation of the amide spectral region indicates that exposure of the sphingosine amide moiety to buffer is greater in the longer chain length DPPC bilayer than in the shorter chain length DMPC bilayer. The thermotropic behavior of the lipid mixtures of C26-CBS at 8 mol % in DMPC or DPPC shows that the glycosphingolipid stabilizes the gel phase of the short chain length bilayer while it destabilizes the long chain length one. Our results further demonstrate that, at this concentration, C26-CBS is completely miscible in DMPC and DPPC in the gel and the liquid crystalline phases. The difference in behavior of C26-CBS in DMPC and DPPC is a consequence of the greater mismatch between the C26 chain length and the bilayer thickness of DPPC relative to DMPC. They may help to understand the deleterious effects of glycosphingolipids with very long chain fatty acids in adrenoleukodystrophy.