We have noted that many histologically and immunohistochemically confirmed pancreatic endocrine tumors show immunophenotypic evidence of acinar cell differentiation, but the clinical relevance of this finding is unknown. We performed this study to evaluate the prevalence and prognostic significance of exocrine differentiation by immunohistochemistry in pancreatic endocrine tumors that do not show morphologic features of acinar cell differentiation. Routinely processed tissue sections from 87 pancreatic endocrine tumors were immunohistochemically stained with monoclonal antibodies against acinar (lipase, chymotrypsin, trypsin) and endocrine cell markers (chromogranin A, neuron-specific enolase, synaptophysin, Leu-7) and for the proliferation-associated peptide Ki67. The degree of staining with each marker was graded on a three-tier scale for acinar markers (grade 0, <5%; grade 1, 5-10%; grade 2, 11-25%; and grade 3, >25%) and on a four-tier scale for endocrine markers (grade 0, <5%; grade 1, 5-25%; grade 2, 26-50%; grade 3, 51-75%; and grade 4, >75%), and the results were correlated with clinical outcome (mean follow-up 53 months). Greater than 75% of the tumor cells stained for chromogranin A, neuron-specific enolase, synaptophysin, and Leu-7 in 100%, 96%, 93%, and 27% of cases, respectively. Overall, 66% of tumors stained positively for at least one acinar cell marker, 31% stained for at least two acinar cell markers, and 13% stained for all three acinar cell markers. Forty-seven percent stained for lipase (23 grade 1, 11 grade 2, seven grade 3), 37% for trypsin (22 grade 1, three grade 2, seven grade 3), and 25% stained for chymotrypsin (13 grade 1, five grade 2, four grade 3). No correlation was noted between the presence or extent of expression of any single or combination of acinar cell markers and clinical outcome. However, higher tumor stage correlated with a poor clinical outcome (p = 0.002), and location in the tail of the pancreas was associated with a longer interval to tumor recurrence (p = 0.03). The presence of synaptophysin (p = 0.03) and Leu-7 expression (p = 0.03) correlated significantly with less aggressive clinical behavior. An association was observed between increased Ki67 labeling and poorer clinical outcome, but this was not statistically significant (p >0.05). In conclusion, immunophenotypic evidence of acinar cell differentiation is common in pancreatic endocrine tumors, but this feature does not have any relevance to clinical prognosis. However, in addition to tumor stage, location in the pancreatic tail and the immunohistochemical expression of synaptophysin and/or Leu-7 may be useful prognostic indicators in patients with these lesions.