Two‐photon excitation fluorescence resonance energy transfer (2P‐FRET) imaging microscopy can provide details of specific protein molecule interactions inside living cells. Fluorophore molecules used for 2P‐FRET imaging have characteristic absorption and emission spectra that introduce spectral cross‐talk (bleed‐through) in the FRET signal that should be removed in the 2P‐FRET images, to establish that FRET has actually occurred and to have a basis for distance estimations. These contaminations in the FRET signal can be corrected using a mathematical algorithm to extract the true FRET signal. Another approach is 2P‐FRET fluorescence lifetime imaging (FLIM). This methodology allows studying the dynamic behavior of protein‐protein interactions in living cells and tissues. 2P‐FRET‐FLIM was used to study the dimerization of the CAATT/enhancer binding protein α (C/EBPα). Results show that the reduction in donor lifetime in the presence of acceptor reveals the dimerization of the protein molecules and also determines more precisely the distance between the donor and acceptor. We describe the development and characterization of the 2P‐FRET‐FLIM imaging system with the Bio‐Rad Radiance2100 confocal/multiphoton microscopy system. Microsc. Res. Tech. 63:72–80, 2004. © 2003 Wiley‐Liss, Inc.