ABSTRACTBy adulthood, the majority of the population is persistently infected with BK polyomavirus (BKPyV). Only a subset of the population, generally transplant recipients on immunosuppressive drugs, will experience disease from BKPyV, but those who do have few treatment options and, frequently, poor outcomes, because to date there are no effective antivirals to treat or approved vaccines to prevent BKPyV. Most studies of BKPyV have been performed on bulk populations of cells, and the dynamics of infection at single-cell resolution have not been explored. As a result, much of our knowledge is based upon the assumption that all cells within a greater population are behaving the same way with respect to infection. The present study examines BKPyV infection on a single-cell level using high-content microscopy to measure and analyze the viral protein large T antigen (TAg), promyelocytic leukemia protein (PML), DNA, and nuclear morphological features. We observed significant heterogeneity among infected cells, within and across time points. We found that the levels of TAg within individual cells did not necessarily increase with time and that cells with the same TAg levels varied in other ways. Overall, high-content, single-cell microscopy is a novel approach to studying BKPyV that enables experimental insight into the heterogenous nature of the infection.IMPORTANCEBK polyomavirus (BKPyV) is a human pathogen that infects nearly everyone by adulthood and persists throughout a person’s life. Only people with significant immune suppression develop disease from the virus, however. Until recently the only practical means of studying many viral infections was to infect a group of cells in the laboratory and measure the outcomes in that group. However, interpreting these bulk population experiments requires the assumption that infection influences all cells within a group similarly. This assumption has not held for multiple viruses tested so far. Our study establishes a novel single-cell microscopy assay for BKPyV infection. Using this assay, we discovered differences among individual infected cells that have not been apparent in bulk population studies. The knowledge gained in this study and the potential for future use demonstrate the power of this assay as a tool for understanding the biology of BKPyV.