Well-differentiated neuroendocrine tumors (WDNET) of the gastrointestinal tract, pancreas, and lung are histologically similar. Thus, predicting the site of origin of a metastasis is not possible on morphologic grounds. Prior immunohistochemical studies of WDNET have yielded conflicting results, and pancreatic and duodenal homeobox factor-1 (PDX-1) has not previously been evaluated in this context. We therefore analyzed the expression of CDX-2, PDX-1, TTF-1, and neuroendocrine secretory protein-55 (NESP-55), a recently described member of the chromogranin family, in primary and metastatic WDNET. In total, 64 gastrointestinal carcinoids (5 stomach; 5 duodenum; 31 ileum; 11 appendix; and 12 rectum); 39 pancreatic endocrine tumors (PET); and 20 pulmonary carcinoid tumors were studied. PET were positive for NESP-55 (16/39) and PDX-1 (11/39); 3/31 also showed heterogeneous positivity for CDX-2. Ileal carcinoids were exclusively positive for CDX-2 (30/31) and negative for all other markers. Appendiceal carcinoids were uniformly positive for CDX-2 (11/11). All rectal carcinoids were negative for CDX-2 and TTF-1; 2/12 were positive for PDX-1, and 1/12 for NESP-55. The gastric and duodenal carcinoids were only positive for PDX-1 (7/10). TTF-1 positivity was confined to pulmonary carcinoids (7/20); 1/20 was positive for NESP-55; and all were negative for CDX-2 and PDX-1. NESP-55 and PDX-1 positivity, in the presence of negative CDX-2 and TTF-1, was 97% specific for PET. The sensitivity and specificity of CDX-2 positivity for predicting an ileal primary, when PDX-1, NESP-55, and TTF-1 were negative, was 97% and 91%, respectively. TTF-1 positivity was confined to pulmonary carcinoids in our study but was present in only about a third of cases. A panel of these 4 markers may be useful in predicting the primary site of metastatic WDNET.