Aldose reductase, the first and rate-limiting enzyme of the polyol pathway, is a target for drug design for the treatment of diabetes complications. The structures of aldose reductase in complex with the cyclic imide inhibitors Fidarestat and Minalrestat were recently determined at ultra-high resolution (Proteins 2004, 55, 805). We have used the detailed structural information revealed at atomic resolution, including the assignment of protonation states for the inhibitors and active site residues, together with molecular modelling and noncovalent mass spectrometry to characterise the type and strength of the interactions between the enzyme and the inhibitors, and to attempt the design of novel potential inhibitors with enhanced binding energies of the complexes. The VC(50) values measured by mass spectrometry (accelerated voltage of ions needed to dissociate 50% of a noncovalent complex in the gas phase) for the aldose reductase inhibitors correlate with the IC(50) values (concentration of inhibitor giving 50% inhibition in solution) and with the electrostatic binding energies calculated between the active site residues Tyr48, His110 and Trp111 and the inhibitors, suggesting that electrostatic interactions play a major role in inhibitor binding. Our molecular modelling and design studies suggest that the replacement of the fluorine atom in Minalrestat's bromo-fluorobenzyl group with nitro, amide and carboxylate functional groups enhanced the predicted net binding energies of the complexes by 16%, 31% and 68%, respectively. When the carbamoyl group of Fidarestat was replaced with a nitro, 4-hydroxyl phenyl and carboxylate functional groups, the predicted net binding energies of the complexes were enhanced by 13%, 34% and 46%, respectively.