The tough corneous layer in the carapace and plastron of hard-shelled turtles derives from the accumulation of keratin-associated beta-proteins (KAbetaPs, formerly called beta-keratins) while these proteins are believed to be absent in soft-shelled turtles. Our bioinformatics and molecular study has instead shown that the epidermis of the soft-shelled turtle Apalone spinifera expresses beta-proteins like or even in higher amount than in the hard-shelled turtle Pseudemys nelsoni. The analysis of a carapace cDNAs library has allowed the identification and characterization of three alpha-keratins of type I and of ten beta-proteins (beta-keratins). The acidic alpha-keratins probably combine with the basic beta-proteins but the high production of beta-proteins in A. spinifera is not prevalent over that of alpha-keratin so that their combination does not determine the formation of hard corneous material. Furthermore the presence of a proline and cisteine in the beta-sheet region of beta-proteins in A. spinifera may be unsuited to form hard masses of corneous material. The higher amount of beta-proteins over alpha-keratins instead occurs in keratinocytes of the hard and inflexible epidermis of P. nelsoni determining the deposition of hard corneous material. The study suggests that the hardness of the corneous layer derives not exclusively from the interactions between alpha-keratins with KAbetaPs but also from the different dynamic of accumulation and loss of corneocytes in the corneous layer of the hard shelled turtles where a prevalent accumulation and piling of corneocytes takes place versus the soft shelled turtle where a rapid turnover of the stratum corneum occurs. J. Exp. Zool. (Mol. Dev. Evol.) 320B: 428–441, 2013. © 2013 Wiley Periodicals, Inc.