A method for the measurement of proton T1ρ relaxation times in unlabeled proteins is described using a variable spin-lock pulse after the initial nonselective 90deg; excitation in a HOHAHA pulse sequence. The experiment is applied to α-bungarotoxin (α-BTX) and its complex with a 25-residue peptide derived from the acetylcholine receptor (AChR) α-subunit. A good correlation between high T1ρ values and increased local motion is revealed. In the free form, toxin residues associated with receptor binding according to the NMR structure of the α-BTX complex with an AChR peptide and the model for α-BTX with the AChR [Samson, A. O., et al. (2002) Neuron 35, 319-332] display high mobility. When the AChR peptide binds, a decrease in the relaxation times and the level of motion of residues involved in binding of the receptor α-subunit is exhibited, while residues implicated in binding γ- and δ-subunits retain their mobility. In addition, the quantitative T1ρ measurements enable us to corroborate the mapping of boundaries of the AChR determinant strongly interacting with the toxin [Samson, A. O., et al. (2001) Biochemistry 40, 5464-5473] and can similarly be applied to other protein complexes in which peptides represent one of the two interacting proteins. The presented method is advantageous because of its simplicity, generality, and time efficiency and paves the way for future investigation of proton relaxation rates in small unlabeled proteins.