The molecular composition of viral particles indicates that a single virion is capable of initiating an infection. However, the majority of viruses that come into contact with cells fails to infect them. Understanding what makes one viral particle more successful than others requires visualizing the infection process directly in living cells, one virion at a time. In this Perspective, we explain how single-virus imaging using fluorescence microscopy can provide answers to unsolved questions in virology. We discuss fluorescent labeling of virus particles, resolution at the subviral and molecular levels, tracking in living cells, and imaging of interactions between viral and host proteins. We end this Perspective with a set of remaining questions in understanding the life cycle of retroviruses and how imaging a single virus can help researchers address these questions. Although we use examples from the HIV field, these methods are of value for the study of other viruses as well.