The process of keratinocyte differentiation was analyzed in the regenerating epidermis of the lizard Anolis carolinensis, where the genes coding for beta-proteins (beta-keratins) are known. The regenerating epidermis forms all epidermal layers found in normal scales (Oberhäutchen-, beta-, mesos-, and alpha-layer). Three specific proteins representing the larger families of beta-proteins, glycine-rich (HgG5, 28% glycine, 3.6% cysteine), glycine-cysteine medium-rich (HgGC10, 13% glycine, 14.5% cysteine), and glycine-cysteine rich (HgGC3, 30.4% glycine, 8.7% cysteine) have been immunolocalized at the ultrastructural level. HgG5 is only present in differentiating beta-cells, a weak or no labeling is observed in Oberhäutchen and is absent in alpha-cells. The protein is located in the pale corneous material forming the compact beta-layer but is absent in mature Oberhäutchen cells. HgGC10 is present among beta-packets in Oberhäutchen and beta-cells but disappears in more compact and electron-pale corneous material. The labeling disappears in mesos-cells and is present with variable intensity in alpha-cells, whereas lacunar and clear-cells are low labeled to unlabeled. HgGC3 is sparse or absent in beta-cells but is lightly present in the darker corneous material of differentiating and mature alpha-cells, lacunar-cells, and clear-cells. The study suggests that while glycine-rich proteins (electron-pale) are specifically used for building the resistant and hydrophobic beta-layer, cysteine–glycine rich proteins (electron-denser) are used to form the pliable corneous material present in the Oberhäutchen and alpha-cells. The differential accumulation of beta-proteins on the alpha-keratin cytoskeleton scaffold and not the alternance of beta- with alpha-keratins allow the differentiation of different epidermal layers. © 2012 Wiley Periodicals, Inc.