We report direct evidence for deprotonation of a lysine side chain buried in the hydrophobic core of a protein, demonstrating heteronuclear H-1-N-15 NMR data on the Lys-66 side chain amine (N zeta) group in the Delta-PHS/V66K variant of staphylococcal nuclease. Previous crystallographic study has shown that the Lys-66 N zeta group is completely buried in the hydrophobic core. On the basis of double and triple resonance experiments, we found that the H-1 zeta and N-15 zeta chemical shifts at pH 8.0 and 6 degrees C for the buried lysine are 0.81 and 23.3 ppm, respectively, which are too abnormal to correspond to the protonated (NH3+) state. Further investigations using a model system suggested that the abnormal H-1 and N-15 chemical shifts represent the deprotonated (NH2) state of the Lys-66 N zeta group. More straightforward evidence for the deprotonation was obtained with 2D F1-H-1-coupled H-1-N-15 heteronuclear correlation experiments. Observed N-15 multiplets clearly indicated that the spin system for the Lys-66 N zeta group is AX(2) (NH2) rather than AX(3) (NH3+) Interestingly, although the amine group is buried in the hydrophobic core, the hydrogen exchange between water and the Lys-66 N zeta group was found to be relatively rapid (93 s(-1) at -1 degrees C), which suggests the presence of a dynamic process such as local unfolding or water penetration. The partial self-decoupling effect on N-15 zeta multiplets due to the rapid hydrogen exchange is also discussed.