Approximately one-half of the amino acid sequence (911 amino acid residues out of 1,880 expected) for bovine plasma fibronectin (cold-insoluble globulin) has been determined. Three types of internal homology were identified, showing that a number of partial gene duplications (multiplications) have occurred during the evolution of this protein. Digestion of fibronectin with plasmin results in major fragments with molecular masses of 29, 170, 23, and 6 kilodaltons (kDal). The NH(2)-terminal 29-kDal fragment consists of 259 residues ordered as five mutually homologous domains (type I homology) with two disulfide bonds in each domain. The 170-kDal fragment shows two to three bands after NaDodSO(4) gel electrophoresis, indicating heterogeneity. This fragment contains the gelatin binding site and the strong heparin binding site present in fibronectin. Digestion of the 170-kDal fragment with chymotrypsin liberates a 45-kDal fragment that also binds to gelatin. This fragment contains at least one domain of type I homology and two domains of type II homology. Further digestion of the 170-kDal fragment with chymotrypsin results in the formation of a 30-kDal fragment that retains the heparin binding activity. This fragment contains sequences constituting type III homology. The 23-kDal fragment consists of 178 residues having three domains of type I homology. The 6-kDal fragment consists of two identical peptides of 26 residues, and these two peptides are linked to each other by two disulfide bonds that form the interchain bridges. Another one of the peptides for which the sequence was determined links the COOH-terminus of the 29-kDal fragment to the NH(2)-terminus of the 170-kDal fragment. This and the fact that the COOH-terminal residue of the 6-kDal fragment is a glutamic acid residue order the four plasmin-digestion fragments as 29-, 170-, 23-, and 6-kDal in the intact fibronectin molecule.