A hydration repulsive force plays an important role in various phenomena. In order to study its temperature dependence, the hydration force of dipalmitoylphosphatidylcholine bilayers was investigated at temperatures below freezing. Using known chemical potential of ice, the strength of the interbilayer repulsive force of dipalmitoylphosphatidylcholine bilayers at subzero temperatures was estimated and X-ray diffraction was used to estimate the interbilayer spacing. An exponentially decaying repulsive force with a decay constant of 0.15 nm was observed. The present subzero temperature data are slightly greater than those measured at temperatures about 20 degreesC.