We describe the implementation of a commercial fluorescence lifetime imaging microscopy (FLIM) instrument used in conjunction with a commercial laser scanning multiphoton microscope. The femtosecond-pulsed near-infrared laser is an ideal excitation source for time-domain fluorescence lifetime measurements. With synchronization from the x-y scanners, fluorescence lifetimes can be acquired on a pixel-by-pixel basis, with high spatial resolution. Multiexponential curve fits for each pixel result in two-dimensional fluorescence resonance energy transfer (FRET) measurements that allow the determination of both proximity of fluorescent FRET pairs, as well as the fraction of FRET pairs close enough for FRET to occur. Experiments are described that characterize this system, as well as commonly used reagents valuable for FRET determinations in biological systems. Constructs of CFP and YFP were generated to demonstrate FRET between this pair of green fluorescent protein (GFP) color variants. The lifetime characteristics of the FRET pair fluorescein and rhodamine, commonly used for immunohistochemistry, were also examined. Finally, these fluorophores were used to demonstrate spatially resolved FRET with senile plaques obtained from transgenic mouse brain. Together these results demonstrate that FLIM allows sensitive measurements of protein–protein interactions on a spatial scale less than 10 nm using commercially available components. © 2003 Society of Photo-Optical Instrumentation Engineers.