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A macroscopic approach has been used to calculate electrostatic energies in complexes of L-lactate dehydrogenase with substrates. The different steps of the catalytic mechanism that have been analysed separately are binding of coenzymes and substrates, protonation of the enzyme, the substrate-induced conformational rearrangement of the protein and the chemical transformation of substrate to product. The contributions of the different amino acid residues to each step have been determined from their electrostatic interaction energies. The overall topology of lactate dehydrogenase protein subunit creates, through helix dipoles, two regions of positive potential. One, in the coenzyme-binding domain, stabilises the negatively charged coenzyme pyrophosphate group. The other stabilises both the anionic substrate binding and the developing negative charge excess on the substrate carbonyl in the transition state. The calculated electrostatic fields are those expected from the roles of amino acid side chains which had been previously suggested from site-directed mutagenesis experiments: that is Asp-53 is important in NADH binding, Arg-171 for pyruvate binding and Arg-109 for transition-state stabilisation. His-195, the proton donor/acceptor in catalysis, is stabilised in the protonated state by both Asp-168 and pyruvate. The closure of the active-site loop sequesters the substrate and the nicotinamide ring of the coenzyme in an internal active-site vacuole. The closure process is calculated to be electrostatically unfavourable due to the buried ionic species. Calculation suggests that favourable hydrophobic effects balance unfavourable electrostatic effects and give the precise balance of energies to drive loop (residues 99–112) movement which generates the catalytically competent species.