Iodinated vinculin, metavinculin and alpha-actinin were used to probe the interaction of these proteins with electrophoretically separated cytoskeletal proteins. Using the gel overlay technique, we detected strong binding of 125I-vinculin and 125I-metavinculin to alpha-actinin, 175 kDa polypeptide, talin, vinculin and metavinculin themselves, and moderate binding to actin. 125I-alpha-actinin was capable of interacting with vinculin and metavinculin. The specific binding of 125-I-alpha-actinin to vinculin and metavinculin immobilized on a polysterene surface was also demonstrated. We suggest that the ability of vinculin and alpha-actinin to form a complex may be realized in microfilament-membrane linkages.