meta-Vinculin and vinculin are closely related proteins that are cytoplasmic components of microfilament-associated cell junctions. This report describes the structural relationship between these two proteins and the genetic basis for tissue-specific expression of meta-vinculin. Analysis of genomic DNA coding for amino acids 676-1066 of vinculin revealed 9 exons spanning an 11.7-kilobase pair region of the genome. In the 4 kilobase pairs of intervening sequence that separates vinculin exons E896-E915 and E916-E984, there is an open reading frame that predicts a sequence homologous to the 68-amino acid peptide specific to porcine meta-vinculin (Gimona, M., Small, J. V., Moeremans, M., Van Damme, J., Puype, M., and Van-dekerckhove, J. (1988) EMBO J. 7, 2329-2334). Analysis of the corresponding cDNA established that chicken meta-vinculin contains a 69-amino acid insertion between residues 915 and 916 of vinculin and that there are no other amino acid sequence differences between chicken vinculin and meta-vinculin. Muscle-specific expression of meta-vinculin occurs by alternative splicing of a transcript produced from a single gene because: all 20 genomic isolates that contain the 3' vinculin exons, also contain the meta-vinculin-specific exon; Southern blots performed at high stringency with exon-specific probes indicate the presence of a single gene; and the 3'-untranslated sequences of vinculin and meta-vinculin cDNAs are identical.