This report describes the isolation and characterization of genomic and cDNA clones which define a subfamily of type I keratins in Xenopus laevis whose expression is restricted to embryonic and larval stages. The XK81 subfamily, named after the prototype cDNA clone DG81, contains four members arranged in two pairs of closely homologous loci; they were named 81A1, A2, B1, and B2. Genomic clones were obtained representing all of these regions. The A1 gene has been completely sequenced together with approximately 1 kb of flanking sequences at each end; this gene corresponds to the previously reported cDNA clone 8128 (Jonas, E., T. D. Sargent, and I. B. Dawid, 1985, Proc. Natl. Acad. Sci. USA, 82:5413-5417). The B2 gene is represented by a partial cDNA clone, DG118. Upstream sequences and about half of the coding regions have been sequenced for the B1 and B2 genes, whereas the A2 locus has been identified on the basis of hybridization data and could be a gene or pseudogene. Genomic Southern blotting indicates that all members of the subfamily have been isolated. The keratin proteins encoded by the B1 and B2 genes are 96% homologous in the central rod domain, whereas A/B gene homology in this region is 81%. During development mRNAs derived from A and B genes accumulate coordinately during gastrula and neurula stages; in the tadpole, 81A mRNA decays rapidly, whereas 81B mRNA shows a second abundance peak, persists for most of tadpole life, and decays by metamorphosis. RNAs derived from the XK81 keratin subfamily are undetectable in the adult, where different type I keratin genes are expressed.