Differential scanning calorimetry (DSC) and X-ray diffraction have been used to study the structure and properties of N-palmitoleoylgalactosylsphingosine (NPoGS; 16:1 galactocerebroside). DSC of fully hydrated NPoGS shows a complex pattern of three endothermic transitions at 35, 39 and 53 degrees C. Using a combination of thermal protocols (varying heating/cooling rates, incubation at different temperatures, etc.), the three ordered chain (gel) phases responsible for the transitions have been isolated; transition I (Tm = 35 degrees C; delta H(I) = 6.3 kcal/mol), transition II (Tm = 39 degrees C; delta HII = 8.6 kcal/mol), and transition III (Tm = 53 degrees C; delta HIII = 12.8 kcal/mol). The gel phases do not interconvert but rather form independently following cooling from the melted chain phase. X-ray diffraction data of the three isolated phases confirm that they all are bilayer structures with different bilayer periodicities (LI, 50.7 A; LII, 51.7 A; LIII = 49.2 A) and different chain packing modes. The LI, LII, and LIII bilayer phases each melt independently to the melted chain L alpha phase. Comparisons with other cerebrosides make it clear that alterations in chain length and chain unsaturation markedly affect the thermotropic behavior of cerebrosides and the metastable and stable phases they are able to form. As with phospholipids, introduction of cis-unsaturation into the N-acyl chain reduces both the chain melting temperature and enthalpy.