Two-dimensional (2D) H-1 NMR spectroscopy was used to study the hapten-binding site of a recombinant antibody F(v) fragment expressed in Escherichia coli. Point mutations of residues in the CDR loops of the F(v) fragment were designed in order to investigate their influence on hapten binding and to make site-specific assignments of aromatic NMR proton signals. Two tyrosines giving NOEs to the ligand 2-phenyloxazolone were identified, residue 33 in CDR1 of the heavy chain and residue 32 in CDR1 of the light chain. The benzyl portion of 2-phenyloxazolone is located between these two residues. The binding site is close to the surface of the F(v) fragment. Comparison with a different anti-2-phenyloxazolone antibody, the crystal structure of which has recently been solved, shows that the general location of the hapten-binding site in both antibodies is similar. However, in the crystallographically solved antibody, the hapten is bound farther from the surface in a pocket created by a short CDR3 loop of the heavy chain. In the binding site identified in the F(v) fragment studied in this report, this space is probably filled by the extra seven residues of the CDR3.