Meta-vinculin, a vinculin-related protein, has been isolated from human uterus smooth muscle. Specific antibodies to meta-vinculin, which distinguish between meta-vinculin and vinculin, were prepared by absorption of anti-meta-vinculin serum on vinculin coupled to nitrocellulose. Meta-vinculin specific antibody demonstrates only smooth and cardiac muscle specificity and is able to cross-react with a small 21-kD fragment of the meta-vinculin polypeptide chain. This antibody does not interact with protease resistant 95-kD core shared by vinculin and meta-vinculin. Meta-vinculin specific antibody was used for the localization of meta-vinculin in smooth and cardiac muscles by the indirect immunofluorescence method. At the light microscopy resolution level it was found that meta-vinculin and vinculin are localized in the same cellular adhesive structures. Meta-vinculin is present in membrane-associated microfilament-bound plaques of smooth muscle, in intercalated discs and costameres of cardiac muscle. In primary culture of smooth muscle cells from human aorta, meta-vinculin and vinculin were found to be present in focal contacts of the cells. During the cultivation of smooth muscle cells, the quantity of meta-vinculin decreased progressively and finally meta-vinculin completely disappeared from the focal contacts. The data show that in smooth and cardiac muscles meta-vinculin could be a structural component of microfilament-membrane attachment sites, defined earlier by the localization of vinculin.