The primary structure of flexilin, an extracellular matrix glycoprotein previously identified in bovine tissues (Lethias, C., Descollonges, Y., Boutillon, M.-M., and Garrone, R. (1996) Matrix Biol. 15, 11-19) was determined by cDNA cloning. The deduced amino acid sequence (4135 residues) reveals that this protein is composed of a succession of peptide motifs characteristic of the tenascin family: an amino-terminal domain containing cysteine residues and heptads of hydrophobic amino acids, 18.5 epidermal growth factor-like repeats, 30 fibronectin type III-like (FNIII) domains, and a carboxyl-terminal fibrinogen-like motif. Sequence analysis indicated that this protein is the bovine orthologue of human tenascin-X. By rotary shadowing, bovine tenascin-X was identified as monomers with a flexible aspect, which are ended by a globule. More FNIII motifs were characterized in the bovine protein than in human tenascin-X. The main difference between the human and bovine tenascin-X is found in the arrangement of the three classes of highly similar FNIII repeat types in the central region of tenascin-X. The bovine FNIII motif b10 exhibits an RGD putative cell attachment site. The functional role of this sequence is corroborated by cell adhesion on purified tenascin-X, which is inhibited by RGD peptides. Moreover, we demonstrate that this RGD site is conserved at the same location in the human molecule.