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Background/purpose As urea is one of the natural moisturizing factor ( NMF) components in the stratum corneum, it has been used in topical products to improve skin conditions. However, the penetration behavior of urea in the skin after application of urea-containing cream has not been determined as there has been no technique with which to measure the urea content in the skin in vivo non-invasively. We therefore applied Raman spectroscopy to evaluate the depth profile of urea content in the skin. We investigated changes in depth profiles of two forms of urea to evaluate the penetration behavior of urea after application of urea-containing cream. Methods Commercially available moisturizing creams F and R in quantities of 2.2-mg/cm2 and containing 20% (w/w) urea were applied to volar forearm skin of six Japanese subjects. Raman spectra of the skin were measured at 2-μm intervals from the skin surface toward the interior using a confocal Raman spectrometer (model 3510 SCA) before and 15, 60, and 120 min after the application of the creams. The amounts of the two forms of urea, urea in water solution and urea in a solid state, were calculated by adding the spectra of solid urea and the cream base to a previously reported algorithm including the spectrum of urea in water solution. Results The characteristic band of urea in water solution was observed at approximately 1004/cm and that of the solid state at approximately 1010/cm in the Raman spectra of the skin after application of either cream. There was more urea in water solution form in the area where cream F was applied than in the area where cream R was applied. There was more urea in a solid state in the area where cream R was applied than in the area where cream F was applied at all depths and measurement times. In particular, there was significantly more urea in a solid state below a depth of 2 μm in the area where cream R was applied than in the area where cream F was applied 15 min after application. Conclusion The present study demonstrated that we can measure both urea forms in the skin after the application of urea-containing creams. The proposed technique would be useful in the evaluation of characteristics of the penetration behavior of urea in the skin after the application of various urea-containing moisturizers. [ABSTRACT FROM AUTHOR]