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The interaction of a new type of hapten, O-carboxymethyl-4-methylumbelliferone (CMU), with its specific IgG antibody and with the Fab fragment of the antibody was studied by 250-MHz proton nuclear magnetic resonance (NMR) and by fluorescence spectroscopy. The hapten fluorescence was quenched by either the IgG or the Fab and by dioxane-water mixtures, and these findings suggest a hydrophobic environment in the active site. Further support for this argument was provided by the positive temperature dependence of the binding constant for the hapten-antibody interaction. There was little quenching of the tryptophan fluorescence of the antibody, however, despite the considerable overlap between the emission spectrum of the antibody and the excitation spectrum of the hapten. This observation suggests that there are no tryptophan residues in the combining site. The number of chemical and physical methods for examining the structure and properties of the antibody binding site have been devised. Fluorescence has provided information regarding the dielectric nature of the binding site, the conformational changes resulting from complex formation, and the strength of the binding interaction (Parker et al., 1967a,b;Werner et al., 1972;Eisen and Siskind, 1964). Nuclear magnetic resonance (NMR) has been useful in studying the binding of ligands to proteins (Dwek, 1973;James, 1975), and the intermolecular nuclear Overhauser effect1 has yielded information on the chemical nature of the groups in the binding sites and their distances from the bound molecule (Bothner-By and Gassend, 1973;Balaram et al., 1972a Balaram et al., ,b, 1973James, 1976). several studies have utilized conventional NMR techniques to investigate the antibody-hapten interaction: Haughland et al. (1967) used the chloride ion probe to study the binding of a dinitrophenylmercurial hapten with a DNP-specific antibody; Burgen et al. (1967) investigated the binding of tetramethylammonium ions to IgG antibody specific for