CONTEXT: Although identification of the primary tumor in patients with metastatic adenocarcinoma has a profound clinical impact, diagnosing the organ of origin is frequently difficult. Because none of the individual immunohistochemical markers used for tissue identification are both site specific and site sensitive, multiple markers are needed to improve the prediction of primary sites. OBJECTIVE: To develop an effective approach to immunohistochemically evaluate metastatic adenocarcinoma for the assignment of a likely primary site of origin. DESIGN: Expression profiles of CDX2, cytokeratin (CK) 7, CK20, thyroid transcription factor 1 (TTF-1), carcinoembryonic antigen (CEA), MUC2, MUC5AC, SMAD4, estrogen receptor (ER), and gross cystic disease fluid protein 15 (GCDFP-15) were generated in adenocarcinomas from 7 primary sites, followed by construction of a decision tree and design of multiple-marker panels. Expression of these markers was evaluated immunohistochemically in 314 primary adenocarcinomas (50 cases each of colorectal, gastric, lung, pancreatic, bile duct, and breast, and 14 cases of ovarian origin) using the tissue array method. Results were validated using 60 cases of metastatic adenocarcinoma with known primaries. RESULTS: Organ-specific immunostaining profiles using multiple markers provided high sensitivity, specificity, and positive predictive value in detecting primary adenocarcinomas, as follows: colorectal, TTF-1-/CDX2+/CK7-/CK20+ or TTF-1-/CDX2+/CK7-/CK20-/(CEA+ or MUC2+); ovarian, CK7+/MUC5AC+/TTF-1-/CDX2-/CEA-/GCDFP-15-; breast, GCDFP-15+/TTF-1-/CDX2-/CK7+/CK20- or ER+/ TTF-1-/CDX2-/CK20-/CEA-/MUC5AC-; lung, TTF-1+ or TTF-1-/CDX2-/CK7+/CK20-/GCDFP-15-/ER-/CEA-/ MUC5AC-; pancreaticobiliary, TTF-1-/CDX2-/CK7+/ CEA+/MUC5AC+; and stomach, TTF-1-/CDX2+/CK7+/ CK20-. Overall, these combined phenotypes correctly predicted the tester samples (metastatic adenocarcinomas with known primaries) in 75% of cases. CONCLUSIONS: Determination of tissue-specific immunostaining profiles is valuable in the diagnostic differentiation of metastatic adenocarcinomas from seven common primary sites and should help to correctly predict the organ of primary tumor origin.