There are no direct means to study class I MHC presentation in human normal or diseased cells. Using CMV-infected human cells and applying novel mAb that mimic T-cell receptor specificity directed toward the immunogenic epitope of the viral pp65 protein presented on HLA-A2 molecules, we directly imaged the dynamics of Ag presentation in infected cells. We demonstrate that following infection large intracellular pools of HLA-A2/pp65 complexes are localized to the Golgi. These HLA-A2/pp65 pools account for the majority of total HLA-A2 molecules in infected cells. Interestingly, these large pools are sequestered inside infected cells and only a small portion of them are exported to the cell surface. Virus-induced class I MHC down-regulation did not affect the intracellular pool of HLA-A2/pp65 complexes. Our data also suggest that proteasome function influences the release of class I complexes to the membrane. We present herein a new and direct molecular tool to study the dynamics of viral Ag presentation that may further elucidate the balance between immune response versus viral escape.