In the course of studies of desmosomes, we found trichohyalin, a 200-kDa protein of the inner root sheath and medulla, in a citric acid-insoluble fraction ("desmosome preparation") from tongue epithelium. Pig tongue epithelium yielded milligram quantities of pure trichohyalin from about 100 g of keratomed epithelium. The protein has an extended shape as determined by gel filtration, ultracentrifugation, and electron microscopy, with a rod domain and a globular domain at one end and overall dimensions of about 85 nm. Crosslinking studies suggest that the protein may be dimeric in solution. The protein is a doublet in some animals but apparently is a single polypeptide of 220 kDa in humans. Immunofluorescence studies showed that it is a major protein of the filiform papillae of the tongue of mammals and is present in isolated cells of the stratum granulosum of some regions of epidermis in a subset of cells containing filaggrin and in the nail matrix. Similarly, in filiform papillae some cells contain granules that stain for both trichohyalin and filaggrin. Immunoblotting confirmed that trichohyalin is present in tongue and epidermis. Polymerase chain reaction with human genomic DNA using oligonucleotide primers based on sheep trichohyalin resulted in synthesis of multiple DNAs, from which a 504-bp fragment was subcloned and sequenced and found to resemble closely the carboxyl terminus of sheep trichohyalin. Studies with antibody to the carboxyl-terminal 14 amino acids of the human sequence show that, whereas the carboxyl-terminal epitope is present only in the stratum granulosum, in epidermis epitopes detected by a monoclonal antibody are demonstrated in both the stratum granulosum and stratum corneum, suggesting that the carboxyl terminus is cleaved in the stratum corneum.