More than half of the known keratin genes (n approximately 50) are expressed in the hair follicle. An in-depth knowledge of their differential expression in this organ will help us to understand the mechanisms of its formation and cycling, and the etiology of inherited hair disorders. Keratin 6hf is a type II keratin recently shown to occur in the companion layer. We cloned the mouse ortholog and characterized its expression in skin and oral mucosa. The mK6hf gene is 9.1 kb long and located in the cluster of type II keratin genes on mouse chromosome 15, between the keratin 6 (mK6alpha/mK6beta) and hair keratin genes. In situ hybridization and protein immunolocalization showed that, in addition to the companion layer, mK6hf is expressed in the upper matrix and medulla of the anagen-stage hair. This distribution is seen for all types of mouse hairs and medullated human hairs. The distribution of keratin 6hf protein in the hair shaft mirrors that of keratin 17, and the observation of reduced levels of keratin 6hf in keratin 17 null hair argues for a direct interaction between them. mK6hf is also expressed in the nail bed epithelium and fungiform papillae of dorsal tongue epithelium. Our findings provide an additional marker for the hair matrix and medulla, and suggest that the cellular precursors for the medulla, cortex, and cuticle compartments are already spatially segregated within the hair matrix. They also have obvious implications for the epithelial alterations associated with defects in keratin 6 genes.