Genetic high-throughput screens have yielded large sets of potential protein-protein interactions now to be verified and further investigated. Here we present a simple assay to directly visualize protein-protein interactions in single living cells. Using a modified lac repressor system, we tethered a fluorescent bait at a chromosomal lac operator array and assayed for co-localization of fluorescent prey fusion proteins. With this fluorescent two-hybrid (F2H) assay we successfully investigated the interaction of proteins from different subcellular compartments including nucleus, cytoplasm and mitochondria. In combination with an S phase marker we also studied the cell cycle dependence of protein-protein interactions. These results indicate that the F2H assay is a powerful tool to investigate protein-protein interactions within their cellular environment and to monitor the response to external stimuli in real-time.