Adenocarcinomas of uncertain origin are a frequent problem for surgical pathologists. To determine the utility of immunostaining for cytokeratin 7 and cytokeratin 20 in the separation of pulmonary adenocarcinomas from colonic adenocarcinomas, we studied routinely processed, formalin-fixed tissue from 151 of these tumors using commercially available monoclonal antibodies and an avidin-biotin immunohistochemical technique. Used alone, neither cytokeratin 7 immunostaining or cytokeratin 20 immunostaining reliably separated these tumors. However, the immunophenotype of cytokeratin 7 positive/cytokeratin 20 negative was seen in 86% of the pulmonary adenocarcinomas, and in 0% of the colonic adenocarcinomas. Conversely, the cytokeratin 7-negative/cytokeratin 20-positive immunophenotype was seen in 77% of the colonic carcinomas, and in 0% of the pulmonary tumors. In conclusion, cytokeratin 7/cytokeratin 20 immunostaining patterns may be helpful in separating pulmonary adenocarcinomas from colonic adenocarcinomas.