We characterized cDNA clones specific for the extracellular matrix glycoprotein undulin. Two sets of cDNA clones were isolated from a human placental lambda gt11 expression library and from a rhabdomyosarcoma cell line encoding two partially identical carboxyl-terminal polypeptides of 843 (Un1) and 443 (Un2) amino acids suggesting differential splicing of a single gene transcript. Northern blot analysis of human rhabdomyosarcoma cell poly (A) RNA with cDNA specific for Un1 identified transcripts of approximately 4.2, 6.5, and 8.5 kilobases, whereas a probe specific for Un2 detected a single mRNA of approximately 5 kilobases. Since a monoclonal antibody that is reactive with a sequence encoded by Un1 and not by Un2 detects the bands considered characteristic for undulin in Western blots, the mRNAs related to Un1 may code for the major part of the undulin molecule. The protein sequences deduced from Un1 and Un2 reveal an amino-terminal differentially spliced von Willebrand factor A domain, characteristic of proteins that interact with interstitial collagens, which is linked to fibronectin-like type III homology units by a unique sequence of 57 amino acids. Whereas Un2 encodes two complete and one incomplete type III homologies followed by a unique acidic carboxyl-terminal domain of 118 amino acids, Un1 codes for seven complete and one truncated type III homologies, followed by a short proline-rich carboxyl-terminal segment of 23 amino acids. Considering the 298 amino acids occurring in identical segments, the 989 different amino acid positions deduced from clones Un1 and Un2 represent an estimated 40% of the overall undulin sequence. In the context of 1) rotary shadowing electron microscopy data showing undulin as a structure composed of nodules that are interconnected by flexible rods of varying size, 2) the presence of three major bands of Mr 270,000, 190,000, and 180,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with 3) common antigenic epitopes and similar peptide maps (Schuppan, D., Cantaluppi, M.C., Becker, J., Veit, A., Bunte, T., Troyer, D., Schuppan, F., Schmid, M., Ackermann, R., and Hahn, E.G. (1990) J. Biol. Chem. 265, 8823-8832), our finding of differentially spliced type III homology units, as found in tenascin and fibronectin, suggests that undulin is another member of the fibronectin-tenascin family of extracellular matrix glycoproteins. Furthermore, as in fibronectin and tenascin, undulin bears an additional subset of interactive domains tailored to specific structural and functional roles in development and differentiation.