Lizard epidermis is made of beta- and alpha-layers. Using Western blot tested antibodies, the ultrastructural immunolocalization of specific keratin-associated beta-proteins in the epidermis of different lizard species reveals that glycine-rich beta-proteins (HgG5) localize in the beta-layer, while glycine–cysteine-medium-rich beta-proteins (HgGC10) are present in oberhautchen and alpha-layers. This suggests a new explanation for the formation of different epidermal layers during the shedding cycle in lepidosaurian epidermis instead of an alternance between beta-keratins and alpha-keratins. It is proposed that different sets of genes coding for specific beta-proteins are activated in keratinocytes during the renewal phase of the shedding cycle. Initially, glycine–cysteine-medium-rich beta-proteins with hydrophilic and elastic properties accumulate over alpha-keratins in the oberhautchen but are replaced in the next cell layer with glycine-rich hydrophobic beta-proteins forming a resistant, stiff, and hydrophobic beta-layer. The synthesis of glycine-rich proteins terminates in mesos and alpha-cells where these proteins are replaced with glycine–cysteine-rich beta-proteins. The pattern of beta-protein deposition onto a scaffold of intermediate filament keratins is typical for keratin-associated proteins and the association between alpha-keratins and specific keratin-associated beta-proteins during the renewal phase of the shedding cycle gives rise to epidermal layers possessing different structural, mechanical, and texture properties.