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The theory that a more thinly keratinized inner foreskin leads to increased HIV-1 susceptibility has been based on relatively little published data. We sought to quantify the keratin thicknesses of the inner and outer foreskin to determine the plausibility of this hypothesis.We took repeated measurements of the keratin layer of 16 adult male foreskins to determine whether differences existed between the inner and outer foreskin.Adult foreskins were collected from consenting donors undergoing elective male circumcision for unknown medical indications in Chicago, Illinois, USA. Specimens were processed, sectioned and stained for keratin using antifilaggrin fluorescent antibodies. Slides stained with hematoxylin and eosin were used as controls and compared with results from previously published studies using this method. Keratin layers were measured in a standardized fashion for each specimen.Comparing our fluorescence-based analysis with previously published immunohistochemical methods revealed that our method was highly accurate for measuring foreskin keratin thickness. There was significant heterogeneity in the keratin thickness of the inner and outer aspects of the male foreskin within and between the different donors. There was no significant difference between the inner and outer foreskin keratin thickness (25.37 ± 12.51 and 20.54 ± 12.51 μm, respectively; P = 0.451).We found no difference between the keratinization of the inner and outer aspects of the adult male foreskin. Keratin layers alone are unlikely to explain why uncircumcised men are at higher risk for HIV infection.