The bacterial replisome is a target for the development of new antibiotics to combat drug resistant strains. The β 2 sliding clamp is an essential component of the replicative machinery, providing a platform for recruitment and function of other replisomal components and ensuring polymerase processivity during DNA replication and repair. A single binding region of the clamp is utilized by its binding partners, which all contain conserved binding motifs. The C-terminal Leu and Phe residues of these motifs are integral to the binding interaction. We acquired three-dimensional structural information on the binding site in β 2 by a study of the binding of modified peptides. Development of a three-dimensional pharmacophore based on the C-terminal dipeptide of the motif enabled identification of compounds that on further development inhibited α-β 2 interaction at low micromolar concentrations. We report the crystal structure of the complex containing one of these inhibitors, a biphenyl oxime, bound to β 2, as a starting point for further inhibitor design. © Published 2011 by the American Chemical Society.