An important but poorly characterized contribution to the thermodynamics of protein-DNA interactions is the loss of entropy that occurs from restricting the conformational freedom of amino acid side chains. The effect of restricting the flexibility of several side chains at a protein-DNA interface may be comparable in many cases to the other factors that determine the binding thermodynamics and may, therefore, play a key role in dictating the binding affinity and/or specificity. Because the entropic contributions, including the presence and influence of side-chain dynamics, are especially difficult to estimate based on structural information, it is important to pursue experimental and theoretical studies that can provide direct information regarding these issues. We report on studies of a model system, the homeodomain/DNA complex, focusing on the Lys50 class of homeodomains where a key lysine residue in position 50 was shown previously to be critical for binding site specificity. NMR methodology was employed for determining the dynamics of lysine side-chain amino groups via 15N relaxation measurements in the Lys50-class homeodomains from the Drosophila protein Bicoid and the human protein Pitx2. In the case of Pitx2, complexes with both a consensus and a nonconsensus DNA binding site were examined. NMR-derived order parameters indicated moderate to substantial conformational freedom for the lysine NH3+ group in the complexes studied. To complement the experimental NMR measurements, molecular dynamics simulations were performed for the consensus complexes to gain further, detailed insights regarding the dynamics of the Lys50 side chain and other important residues in the protein-DNA interface.