O-tert-Butyltyrosine (Tby) is an unnatural amino acid that can be site-specifically incorporated into proteins using established orthogonal aminoacyl-tRNA synthetase/tRNA systems. Here we show that the tert-butyl group presents an outstanding NMR tag that can readily be observed in one-dimensional H-1 NMR spectra without any isotope labeling. Owing to rapid bond rotations and the chemical equivalence of the protons of a solvent-exposed tert-butyl group from Tby, the singlet resonance from the tert-butyl group generates an easily detectable narrow signal in a spectral region with limited overlap with other methyl resonances. The potential of the tert-butyl H-1 NMR signal in protein research is illustrated by the observation and assignment of two resonances in the Bacillus stearothermophilus DnaB hexamer (320 kDa), demonstrating that this protein preferentially assumes a 3-fold rather than 6-fold symmetry in solution, and by the quantitative measurement of the submicromolar dissociation constant K-d (0.2 mu M) of the complex between glutamate and the Escherichia coli aspartate/glutamate binding protein (DEBP, 32 kDa). The outstanding signal height of the H-1 NMR signal of the Tby tert-butyl group allows K-d measurements using less concentrated protein solutions than usual, providing access to Kd values 1 order of magnitude lower than established NMR methods that employ direct protein detection for Kd measurements.