The pituitary-specific transcription factor Pit-1 forms dimers when interacting with specific DNA elements and has been shown to associate with several other nuclear proteins. Recently, techniques have become available that allow visualization of protein-protein interactions as they occur in single living cells. In this study, the technique of fluorescence resonance energy transfer (FRET) microscopy was used to visualize the physical interactions of Pit-1 proteins fused to spectral variants of the jellyfish green fluorescent protein (GFP) that emit green or blue light [blue fluorescent protein (BFP)]. An optimized imaging system was used to discriminate fluorescence signals from single cells coexpressing the BFP- and GFP-fusion proteins, and the contribution of spectral overlap to background fluorescence detected in the FRET images was established. Energy transfer signals from living cells expressing a fusion protein in which GFP was tethered to BFP by short protein linker was used to demonstrate acquisition of FRET signals. Genetic vectors encoding GFP- and BFP-Pit-1 proteins were prepared, and biological function of the fusion proteins was confirmed. FRET microscopy of HeLa cells coexpressing the GFP- and BFP-Pit-1 demonstrated energy transfer, which required the two fluorophores to be separated by less than 100 A. Biochemical studies previously demonstrated that Pit-1 physically interacts with both c-Ets-1 and the estrogen receptor. FRET imaging of cells coexpressing BFP-Pit-1 and GFP-Ets-1 demonstrated energy transfer between these fusion proteins, a result consistent with their association in the nucleus of these living cells. In contrast, there was no evidence for energy transfer between the BFP-Pit-1 and an estrogen receptor-GFP fusion proteins. It is likely that the FRET imaging approach described here can be applied to many different protein-partner pairs in a variety of cellular contexts.