The immunohistochemical expression and localization of monoclonal antibodies to carcinoembryonic antigen (CEA) and human alveolar macrophage (HAM-56) were evaluated in primary ovarian and metastatic gastrointestinal (GI) carcinomas. Immunohistochemistry was performed using an avidin-biotin-peroxidase complex method with capillary gap technology on formalin-fixed, paraffin-embedded tissues from 41 primary ovarian epithelial neoplasms, 17 metastatic gastrointestinal malignancies, and 10 tumors of uncertain primary origin. Overall, immunostaining for HAM-56 was positive in 35 (85%) ovarian epithelial neoplasms compared with only two (12%) gastrointestinal cancers. Carcinoembryonic antigen was positive in 16 (39%) ovarian versus 13 (76%) GI tumors. Of the primary ovarian neoplasms, 22 were positive for HAM-56 only, 13 were positive for both HAM-56 and CEA, three were positive for CEA only (all mucinous neoplasms), and three were negative for both. Of the primary GI neoplasms, 12 were positive for CEA only (including all eight colon cancers), one was positive for both HAM-56 and CEA, one was positive for HAM-56 only, and three were negative for both. Of the 10 neoplasms of unknown origin at initial presentation, six were positive for HAM-56 only, three were positive for CEA only, none was positive for both HAM-56 and CEA, and one was negative for both. Only three of these 10 neoplasms remained of indeterminate origin after pathological review and clinical follow-up. When positive, CEA was usually strong and generalized in GI cancers but weak and focal in ovarian neoplasms. The HAM-56 positivity in ovarian neoplasms was typically focal and largely limited to areas with glandular or papillary differentiation with apical linear accentuation. We conclude that an immunohistochemical panel using both HAM-56 (Enzo Diagnostics, Syosset, NY) and CEA monoclonal antibodies is helpful in differentiating primary ovarian neoplasms from metastatic gastrointestinal malignancies, and in evaluating metastatic adenocarcinoma of unknown primary site.