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Synchrotron X-ray diffraction was employed to evaluate the effect of ethanol and L-menthol on lipid arrangements in the stratum corneum of hairless rats. Two sharp diffractions (S=2.40 and S=2.67, corresponding to spacing of 0.417 nm and 0.374 nm respectively) were observed on the broad hump peak derived from soft keratin. To assist in understanding the effects of treatment with ethanol and L-menthol, an abundance ration of lipid hydrocarbon chain packing index (RH/O) was defined as RH/O=(Peak area at S=2.40 nm−1)/(Peak area at S=2.67 nm−1). When ethanol was applied to the stratum corneum the intensities of diffraction peaks declined slightly. The RH/O values observed were not affected by variations in ethanol concentrations in the range 0–40% (w/w). The RH/O values did not change even when treatment with ethanol (40%, w/w) was extended to 8 h. These results suggested that lipid arrangements in the stratum corneum were not affected by ethanol. On the other hand, exposure of the stratum corneum to 2% (w/w) L-menthol caused a significant decrease in RH/O value. It was shown that L-menthol was dispersed through the stratum corneum, intruded mainly into hexagonal hydrocarbon chain packing, and disrupted the regular organization of these structures.