AKR/J mice display a hair interior defect (hid) phenotype for which the molecular basis is unknown. To investigate the application of hair-shaft proteomics to the study of such diseases, pelage from AKR/J and two other mouse strains without this defect was analyzed by shotgun proteomics. The results permitted the identification of 111 proteins from tryptic digests of total hair from AKR/J-hid/hid mice, which were predominantly keratins (Krts) and Krt-associated proteins (Krtaps). From the non-solubilizable (crosslinked) fraction of the hair remaining after extensive detergent extraction, 58 proteins were identified. The majority were Krts and Krtaps, but junctional and other membrane proteins, cytoplasmic proteins, and histones were also identified. The results indicate the incorporation of a multitude of proteins into highly crosslinked material. Comparison of unique peptides generated among hair samples from AKR/J-hid/hid, FVB/NJ+/+, and LP/J+/+ mice indicated that these inbred strains could be distinguished by their proteomic patterns. Transmission electron microscopy after mild treatment in detergent and reducing agent permitted the visualization of projections of cortex cells, with characteristic filament patterns, into adjoining medulla cells. Hair shafts from AKR/J mice were deficient in these projections and also exhibited relatively low levels of trichohyalin, a possible contributor to or marker for the hid phenotype.