Differential scanning calorimetry (DSC) and fluorescence spectroscopy are useful techniques in determining the threshold value for the onset of interdigitation in distearoylphosphatidylcholine (DSPC) multilayers. In both DSPC and DSPC/2 mol% cholesterol systems, the threshold value was found to be 4% ethanol. DSC results showed the biphasic effect with the disappearance of the pretransition below the threshold value and shoulder peaks on cooling scans above the threshold value. 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to detect the great decrease in intensity as an indicator for interdigitation. Cholesterol lowered and broadened the transition temperatures of the phosphatidylcholine. 2 mol% cholesterol did not have much effect in the induction of interdigitated phase in DSPC.