We describe a monoclonal antibody (anti-FC-1) that inhibits the attachment of chicken embryo fibroblasts to artificial substrates. In the immunofluorescence, anti-FC-1 also stained the focal contacts of the fibroblasts, organelles suggested to be involved in cell-substrate adhesion. This antibody was absorbed on the surface of intact fibroblasts, and its action was blocked by cell extracts with nonionic detergents, indicating that the antigen (FC-1) is a surface membrane protein. Triton-extracted FC-1 exhibited a molecular weight of approximately 500,000, and a 60,000 dalton protein was immunoprecipitated from 35S-methionine-labeled cells. We have thus characterized a new protein at the exterior side of fibroblast's focal contacts. To determine to what degree this protein mediates the adhesion of the cells to the substrate, and whether it also interacts with the cytoskeletal elements on the cytoplasmic side, will require further investigation.