Small proteins termed β-keratins constitute the hard corneous material of reptilian scales. In order to study the cell site of synthesis of β-keratin, an antiserum against a lizard β-keratin of 15–16 kDa has been produced. The antiserum recognizes β-cells of lizard epidermis and labels β-keratin filaments using immunocytochemistry and immunoblotting. In situ hybridization using a cDNA-probe for a lizard β-keratin mRNA labels β-cells of the regenerating and embryonic epidermis of lizard. The mRNA is localized free in the cytoplasm or is associated with keratin filaments of β-cells. The immunolabeling and in situ labeling suggest that synthesis and accumulation of β-keratin are closely associated. Nuclear localization of the cDNA probe suggests that the primary transcript is similar to the cytoplasmic mRNA coding for the protein. The latter comprises a glycine-proline-rich protein of 15.5 kDa that contains 163 amino acids, in which the central amino acid region is similar to that of chick claw/feather while the head and tail regions resemble glycine-tyrosine-rich proteins of mammalian hairs. This is also confirmed by phylogenetic analysis comparing reptilian glycine-rich proteins with cytokeratins, hair keratin-associated proteins, and claw/feather keratins. It is suggested that different small glycine-rich proteins evolved from progenitor proteins present in basic (reptilian) amniotes. The evolution of these proteins originated glycine-rich proteins in scales, claws, beak of reptiles and birds, and in feathers. Some evidence suggests that at least some proteins contained within β-keratin filaments are rich in glycine and resemble some keratin-associated proteins present in mammalian corneous derivatives. It is suggested that glycine-rich proteins with the chemical composition, immunological characteristics, and molecular weight of β-keratins may represent the reptilian counterpart of keratin-associated proteins present in hairs, nails, hooves, and horns of mammals. These small proteins produce a hard type of corneous material due to their dense packing among cytokeratin filaments. Anat Rec Part A, 288A:734–752, 2006. © 2006 Wiley-Liss, Inc.