Single-molecule Förster resonance energy transfer (FRET) measurements with phosphoglycerate kinase from yeast were performed at different concentrations of guanidine hydrochloride. From these steady-state measurements we obtained FRET efficiency histograms characterizing structural properties of individual proteins at different stages between the native and the fully unfolded state. Native proteins exhibit a slightly more expanded structure under buffer conditions without denaturant as compared to conditions with denaturant. At 0.5 M GndHCl an unfolded state population that exhibits a significantly expanded structure as compared to the native state, emerges. The unfolded state is characterized by a pronounced broadening of the efficiency distribution, which indicates a large structural and/or dynamical heterogeneity within the population. At high denaturant concentrations, well above the unfolding transition at C1/2∼0.7 M, we observe a progressive expansion of the protein structure, namely globule–coil transition.