The β-keratins constitute the hard epidermis and adhesive setae of gecko lizards. Nucleotide and amino acid sequences of β-keratins in epidermis of gecko lizards were cloned from mRNAs. Specific oligonucleotides were used to amplify by 3′- and 5′-rapid amplification of cDNA ends analyses five specific gecko β-keratin cDNA sequences. The cDNA coding sequences encoded putative glycine-proline-serine–rich proteins of 16.8–18 kDa containing 169–191 amino acids, especially 17.8–23% glycine, 8.4–14.8% proline, 14.2–18.1% serine. Glycine-rich repeats are localized toward the initial and end regions of the protein, while a central region, rich in proline, has a strand conformation (β-pleated fold) likely responsible for the formation of β-keratin filaments. It shows high homology with a core region of other lizard keratins, avian scale, and feather keratins. Northern blotting and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis show a higher β-keratin gene expression in regenerating epidermis compared with normal epidermis. In situ hybridization confirms that mRNAs for these proteins are expressed in cells of the differentiating oberhautchen cells and β-cells. Expression in adhesive setae of climbing lamellae was shown by RT-PCR. Southern blotting analysis revealed that the proteins are encoded by a multigene family. PCR analysis showed that the genes are presumably located in tandem along the DNA and are transcribed from the same DNA strand like in avian β-keratins. Developmental Dynamics 236:374–388, 2007. © 2006 Wiley-Liss, Inc.