Studies on the regulation of viral transcription upon infection of the target cells have provided important information on the viral and host factors that influence pathogenesis. However, these studies have been limited so far to steady-state analysis of gene expression. Here we report an image based photon-counting method that allows real-time quantitative imaging of viral gene expression in infected single cells. Employing an HIV-1 vector bearing the firefly luciferase reporter gene, we exploited a single cell photon imaging methodology (a customized and highly sensitive imaging microscope) to measure viral gene expression following integration into a host genome in situ. Our approach reveals real-time dynamics of viral gene expression in living HIV natural target cells (primary human CD4 T cells and macrophages), and promises itself as a powerful tool for quantitative studies on a wide variety of virus-host cell interactions.