The enzyme-bound conformation of C-lactose, an Escherichia coli beta-galactosidase inhibitor has been determined by NMR spectroscopy. It is demonstrated that the enzyme selects a high-energy conformation of this closely related structural analogue of the natural substrate, lactose. In addition, a molecular modeling protocol has been performed in order to obtain a detailed three-dimensional structure of the complex that can explain, in structural terms, the role that the key amino acid residues play in the catalytic mechanism. The implications of the recognition of a high-energy conformation of the analogue are also outlined.