Background: The use of ultrastructural analysis in the diagnostic work-up of histologic specimens has been well studied but less is known about the utility of electron microscopy (EM) in cytopathology. Design: 149,006 non-gynecologic cytology cases at the Massachusetts General Hospital between the years 1993 and 2006 were searched to identify those in which material had been submitted for EM. Cytologic and EM diagnoses were correlated with available histologic diagnoses. The results were put into one of three categories: confirmatory, diagnostic, or insufficient material for diagnosis (IMFD). Results: Material for EM was obtained from 178 cytology cases that included 131 fine-needle aspirates (FNA) and 47 exfoliative specimens. EM provided additional diagnostic information beyond that offered by cytologic examination alone in 32% of cases, and in 48% of cases EM confirmed the cytologic findings. Insufficient material and discrepant results were noted for EM evaluation in 19% of cases and in 1% cases respectively. EM was most useful when applied to FNAs for subclassifying tumors as epithelial or mesenchymal (45.6%), for the diagnosis of non-neoplastic processes (15.7%) such as alveolar proteinosis and amyloidosis, and for the identification of microorganisms (12.2%). In our study, although EM was infrequently applied to exfoliative specimens to distinguish mesothelioma from adenocarcinoma, it proved to be very useful in this setting. Conclusion: When adequate material is obtained, EM can contribute significantly to the evaluation of both FNA and exfoliative cytology cases, including the diagnosis and subclassification of epithelial and mesenchymal tumors, non-neoplastic processes, and the identification of microorganisms.</.