We report a protein-fragment complementation assay (PCA) based on the engineered Deinococcus radiodurans infrared fluorescent protein IFP1.4. Unlike previous fluorescent protein PCAs, the IFP PCA is reversible, allowing analysis of spatiotemporal dynamics of hormone-induced signaling complexes in living yeast and mammalian cells at nanometer resolution. The inherently low background of infrared fluorescence permitted detection of subcellular reorganization of a signaling complex expressed at low abundance.