Metavinculin is a higher mol. wt variant of vinculin expressed only in muscle tissue. Using amino acid sequencing methods on the intact molecules and their proteolytic subfragments, together with a polyclonal antibody specific only for metavinculin from porcine stomach, we have been able to identify and sequence the difference peptide in the porcine metavinculin molecule. By alignment with the complete sequence of chick fibroblast vinculin (communicated by G.J. Price, P. Jones, M.D. Davison, R. Bendori, S. Griffiths, B. Patel, B. Geiger and D.R. Critchley, prior to publication) the exact location of the insert could be determined. In porcine metavinculin, this insert lies between the 90-kd protease-resistant amino-terminal core and the carboxy terminus of the molecule. It contains 68 amino acids and is flanked by KWSSK sequences, one of which is present in vinculin. The identity of the mapped vinculin and metavinculin sequences outside this difference peptide is consistent with the two proteins arising via alternative splicing at the mRNA level. The lack of reactivity of the porcine metavinculin antibody with metavinculin from chicken as well as the finding of different proteolytic cleavage sites in avian metavinculin indicate a species-specific amino acid sequence in the difference piece of the metavinculin molecule.