The thermotropic behavior of the hydrated bilayers formed by novel glycosylceramides containing an anomeric galactose, a saturated long acyl chain (cerotoyl, C-26), and a phytosphingosine base, alpha- and beta-GalpCer, was investigated by means of differential scanning calorimetry (DSC) and X-ray diffraction measurements. DSC curves for hydrated alpha-GalpCer showed an endothermic transition from metastable gel to liquid crystalline states at 72.8 degrees C (Delta H = 12.0 kcal/mol) and exothermic transition of the reverse change at 66.3 degrees C (Delta H = -10.9 kcal/mol) at the heating/cooling rate of 1 degrees C/min. The metastable gel of alpha-GalpCer was remarkably stable in contrast with beta-GalpCer or the typical animal beta-galactosylceramide containing a long acyl chain. Annealing at 37 degrees C for 48 h completely converted the metastable gel of alpha-GalpCer into a stable gel, which was directly transformed to a liquid crystal (LC) at 74.9 degrees C with a a-fold larger endothermic peak (Delta H =-23.8 kcal/mol). Cooling of the LC state brought about the transition into the metastable gel. X-ray diffraction measurements showed that both metastable and stable gels were lamellar structures. The diffraction patterns also suggested that the stable gel had a bilayer arrangement of higher crystallinity than the metastable gel. However, the metastable gel of beta-GalpCer was rapidly converted to the stable gel: Upon heating, the stable gel was directly transformed to the LC state at 75.8 degrees C(Delta H = 19.0 kcal/mol), and cooling of the LC state led to successive transitions of the LC to the metastable gel at 71.0 degrees C and of the metastable gel to the stable gel at 64.8 degrees C. The stable gel gave sharp reflections at 1/4.66 and 1/4.06 Angstrom(-1). DSC findings also showed that at least 30 mol % of alpha-GalpCer was intermixing and the lower fraction of beta-GalpCer was miscible with DPPC in the gel state. The less ordered metastable gel of alpha-GalpCer was assumed to be involved in the better miscibility with DPPC.