We have previously identified two genes (EMS1 and PRAD1/cyclin D1) in the chromosome 11q13 region that are frequently coamplified and overexpressed in human breast cancer and in squamous cell carcinomas of the head and neck (E. Schuuring, E. Verhoeven, W.J. Mooi, and R.J.A.M. Michalides, Oncogene 7:355-361, 1992). We now report on the characterization of the 80/85-kDa protein that is encoded by the EMS1 gene. Amino acid sequence comparison shows a high homology (85%) to a chicken protein that was recently identified as a substrate for the src oncogene (H. Wu, A.B. Reynolds, S.B. Kanner, R.R. Vines, and J.T. Parsons, Mol. Cell. Biol. 11:5113-5124, 1991). Immunocytochemistry reveals that in epithelial cells, the human EMS1 protein is localized mainly in the cytoplasm and, to a very low extent, in protruding leading lamellae of the cell. However, in carcinoma cells that constitutively overexpress the protein as a result of amplification of the EMS1 gene, the protein, except in cytoplasm, accumulates in the podosome-like adherens junctions associated with the cell-substratum contact sites. The protein was not found in intercellular adherens junctions. Our findings, and the previously reported observations in src-transformed chicken embryo fibroblasts, suggest that the EMS1 protein is involved in regulating the interactions between components of adherens-type junctions. Since amplification of the 11q13 region has been associated with an enhanced invasive potential of these tumors, overexpression and concomitant accumulation of the EMS1 protein in the cell-substratum contact sites might, therefore, contribute to the invasive potential of these tumor cells.