Neuroendocrine secretory protein-55 (NESP-55), the latest addition to the chromogranin family, is a product of a genomically imprinted gene transcribed exclusively from the maternal allele. Initial studies have shown it to have a less widespread distribution than that of chromogranin A in normal tissues. It has also been suggested that NESP-55 may be a marker of neuroendocrine tumors differentiating toward the adrenal chromaffin and pancreatic islet cells. Metastatic gastrointestinal and pulmonary carcinoids may occasionally be difficult to distinguish from pancreatic endocrine tumors (PETs) and pheochromocytomas on morphologic grounds alone. We studied neuroendocrine tumors from these sites to see if NESP-55 expression could reliably discriminate pulmonary and gastrointestinal carcinoids from neuroendocrine tumors arising in the pancreas or the adrenal medulla. Sixty-three neuroendocrine tumors positive for one or more immunohistochemical marker of neuroendocrine differentiation (chromogranin A, chromogranin B, synaptophysin, secretogranin II, neuron-specific enolase) were selected for the study and consisted of 34 typical carcinoids (15 pulmonary, 11 ileal, 4 gastric, and 4 rectal), 19 PETs, and 10 pheochromocytomas (4 sporadic, 3 MEN-2, 2 neurofibromatosis type 1, and 1 VHL). All cases were stained for NESP-55 after microwave antigen retrieval using a rabbit polyclonal antibody at a dilution of 1:1000. Sections of normal adrenal medulla were used as positive controls for NESP-55 staining. Negative controls consisted of omission of primary antibody and replacement with normal rabbit serum at an equivalent concentration. NESP-55 immunoreactivity was seen as brown finely granular cytoplasmic staining with prominent perinuclear accentuation. All gastric and ileal carcinoids studied were completely negative for NESP-55. One of four rectal and 1 of 15 pulmonary carcinoids showed focal positivity for it in less than 5% of tumor cells. In contrast, all 10 pheochromocytomas and 14 of 19 PETs showed strong immunohistochemical staining in a variable proportion of tumor cells. Diffuse positivity (>75% of tumor cells) was seen in 6 of 14 PETs and 8 of 10 pheochromocytomas. Our results indicate that, in contrast to the other granins, NESP-55 reactivity is restricted to endocrine tumors of the pancreas and the adrenal medulla. Immunohistochemical expression of NESP-55 may thus be useful in assigning a pancreatic or adrenal origin to metastatic endocrine tumors of unknown origin.