Human immunodeficiency virus type 1 (HIV-1) protease, being responsible for the cleavage of the viral polyproteins gag and gag-pol , is an essential component in the processing of viral proteins encoded in the HIV viral genome. The present work utilizes site-directed spin labeling (SDSL) and double electron-electron resonance (DEER) spectroscopy to characterize flap conformations in HIV-1 protease in an inhibited and uninhibited form. Because HIV protease is a homodimer, incorporation of the labeling probe at K55C yields a spin label pair within the protease, suitable for distance measurements by pulsed techniques. DEER experiments were performed with and without protease inhibitors to determine distances between residues 55-55' within the flap region of the protease. Analysis of the EPR results demonstrates that the flap region spans a continuous, rather than segmental, range of motion in its uninhibited state. Upon addition of inhibitor, the distance between spin labels shortens by similar to 3-4 angstrom (regardless of spin label choice), indicating a closing of the flaps. More importantly, these results characterize the range of flap opening in an uninhibited form, showing distances that span 26-48 angstrom, indicative of a broad range of possible conformations.