AbstractIntroduction: Polyomavirus infections are common in the general (adult) population with a reported prevalence of more than 80%. Polyomavirus can infect urothelial carcinoma and change the morphology of these malignant cells, as is shown in this paper.Material and Methods: An eighty year old Hispanic male was referred to the urology clinic for diagnosis and treatment. The submitted voided urine sample was concentrated, processed, and a SurePath preparation was made.Results and Discussion: The Papanicolaou‐stained slides contained single cells with very large hyperchromatic nuclei with a glassy appearance in addition to atypical neoplastic cells. The single cells with enlarged nuclei proved to stain positive for Simian virus 40 (SV40) antigen. The nuclei of these cells were 2 to 4 times larger than that of the surrounding noninfected atypical cells. These findings were confirmed on histologic sections prepared from tissue biopsy.Conclusion: In conclusion we report that it is difficult to distinguish benign polyomavirus infected cells from their malignant counterparts in cytology but not in histology. Diagn. Cytopathol. 2010. © 2010 Wiley‐Liss, Inc.