Protein-protein interactions play a pivotal role in coordinating many cellular 
processes. Determination of subcellular localization of interacting proteins and 
visualization of dynamic interactions in living cells are crucial to elucidate 
cellular functions of proteins. Using fluorescent proteins, we previously 
developed a bimolecular fluorescence complementation (BiFC) assay and a 
multicolor BiFC assay to visualize protein-protein interactions in living cells. 
However, the sensitivity of chromophore maturation of enhanced yellow 
fluorescent protein (YFP) to higher temperatures requires preincubation at lower 
temperatures prior to visualizing the BiFC signal. This could potentially limit 
their applications for the study of many signaling molecules. Here we report the 
identification of new fluorescent protein fragments derived from Venus and 
Cerulean for BiFC and multicolor BiFC assays under physiological culture 
conditions. More importantly, the newly identified combinations exhibit a 
13-fold higher BiFC efficiency than originally identified fragments derived from 
YFP. Furthermore, the use of new combinations reduces the amount of plasmid 
required for transfection and shortens the incubation time, leading to a 2-fold 
increase in specific BiFC signals. These newly identified fluorescent protein 
fragments will facilitate the study of protein-protein interactions in living 
cells and whole animals under physiological conditions.