To answer the fundamental question of why fluorescent proteins are fluorescent, we performed fluorescence lifetime measurements of enhanced yellow fluorescent protein (eYFP), its 19 different Y145 mutants, and its 19 different H148 mutants. The lifetimes of the Y145 mutants varied drastically over a wide range of 3.4 ns to 82 ps, whereas the variation in the lifetime of the H148 mutants was relatively small. This result demonstrates that the single 145th side chain can control the fluorescence efficiency of eYFP. Furthermore, it was found that the steric effect of the 145th side chain is responsible for controlling the fluorescence efficiency. (C) 2014 Elsevier B.V. All rights reserved.