The hair follicle provides an excellent system in which to study growth and differentiation. Hair keratins are useful tissue-specific molecular markers for these events. By comparing a second mouse Type I hair keratin cDNA clone, MHKA-2, with our previously described MHKA-1, we have been able to contrast the nucleotide sequences and corresponding deduced amino acid sequences of the smallest (mHa4) and the largest (mHa1) major Type I hair keratins. Both nucleotide sequences and both deduced amino acid sequences share high identity but have distinct segments suitable for generation of specific molecular probes. Comparison of amino acid sequences adjacent to the central helical domains has demonstrated homologous subdomains, designated H1 and H2, in the Type I hair keratin nonhelical termini. Although there is only 56% amino acid identity in the carboxy-terminal nonhelical domains, a common sequence, T-------CGPC----R, has been identified in this domain, suggesting a possible common functional role for this portion of the molecule. In addition, it appears that mHa4 may differ in part from mHa1 by deletion of a segment between the H2 subdomain and the conserved sequence. Staining of mouse and human hair follicles with AmHa1, a monospecific polyclonal antibody for mHa1, and AE13, an antibody specific for all Type I hair keratins, suggests differential expression of individual Type I hair keratins in both species. This supports our hypothesis that distinct functional requirements are satisfied by the multiplicity of hair keratins.