Single-molecule fluorescence experiments on the ClpA ATPase component of the ClpAP protease demonstrate that ClpA binds its peptide substrates in discrete high- and low-affinity conformations. These conformations correspond to different states in the catalytic cycle of ATP hydrolysis. On the basis of these observations, we propose that translocation of substrates through the central pore of ClpA is driven by a switch between high- and low-affinity states.