The ClpXP ATPase-protease complex is a key element of the protein quality control machinery in the cell. ClpX consists of a zinc-binding domain (ZBD) that forms dimers and a AAA(+) domain that arranges into a hexamer in an ATP-dependent manner. Here, we report the binding site of the ClpX substrate lambda phage protein O (lambda O) on ZBD(2) in ClpX using NMR and mutagenesis analysis. lambda O protein was found to interact with a hydrophobic patch on the larger surface of ZBD(2). The affinity of lambda O toward ZBD(2) was investigated using a quantitative optical biosensor method of dual polarization interferometry. The data suggest overlapping binding sites of lambda O and the ClpX cofactor SspB on the ZBD(2). Interestingly, a single key mutation in ZBD was found to enhance the ClpXP-dependent degradation of lambda O.