The effects of solutes on spin-label mobility and protein conformation have been investigated with X-band continuous-wave and pulsed electron paramagnetic resonance (EPR) spectroscopy for spin labels attached to an aqueous-exposed site in the beta-hairpin flap region of HIV-1 protease. Specifically, we examined the effects of glycerol, sucrose, PEG3000, and Ficoll400 for four commonly used nitroxide spin labels and found that the largest perturbations to the EPR line shapes occur for solutions containing PEG3000 and glycerol. From comparisons of the spectral line shapes and distance distribution profiles of spin-labeled HIV-1 protease with and without inhibitor, it was concluded that solutes such as glycerol and PEG3000 alter the line shapes of the spin label in the beta-hairpin flaps of HIV-1 PR by modulation of spin-label mobility through changes in preferential interactions with the solutes. It is noteworthy that the high osmolality of the 40% glycerol solution did not alter the conformation of the flaps as determined from pulsed EPR distance measurements.