We have prepd. a bichromophoric crosslinked variant of GCN4-P1 for single-mol. fluorescence energy transfer expts. (GCN4-Pf). The folding and unfolding fluctuations of single GCN4-Pf mols. are measured in a two-channel confocal microscope with which donor and acceptor fluorescence trajectories are measured simultaneously. The energy transfer efficiency is thereby detd. and its probability distributions as a function of added denaturant [urea] are calcd. The distributions indicate that single mol. GCN4-Pf is in dynamic folding equil. with the position of the equil. being altered by the concn. of urea.