The quaternary ammonium binding locus in the active site of mammalian acetylcholinesterase is subtended by the side chains of Trp86, Tyr133, Glu202, and Tyr337. Linear free-energy relationships define the interactions involved in molecular recognition by mouse acetylcholinesterase of the quaternary ammonium moiety of ligands. For substrates CH3C(=O)XCH2CH2Y [X = O, Y = CHMe2, or CH2CH3; X = S, Y = H, NH+Me2, or N+Me3 ] and trifluoroacetophenone transition state analogue inhibitors m-YC6H4C(=O)CF3 [Y = H, Me, Et, iPr, tBu, CF3, NH2, NO2, NMe2, or N+Me3], log(k(cat)/K-m) and pK(i) depend linearly on the molar refractivity, but not the hydrophobicity, of the substituents Y. These correlations indicate that, in the acylation stage of catalysis, interactions in the quaternary ammonium binding locus stabilize the tetrahedral intermediate (as modeled by transition state analogue affinity) by (5 x 10(5))-fold (Delta G(TI) = -32.5 kJ mol(-1)) and the transition state by (2 x 10(4))-fold (Delta Delta G(double dagger) = -24.5 kJ mol(-1)). To evaluate the contribution of cation-pi interactions, Trp86 was converted into Tyr, Phe, and Ala by site-specific mutagenesis. For this set of enzymes, a linear free-energy relationship is observed between the pK(i) values for inhibitions by the respective neutral and cationic transition state analogue inhibitors, m-tert-butyltrifluoroacetophenone and m-(N,N,N-trimethylammonio)-trifluoroacetophenone, which indicates that the free energy released on interaction of the: quaternary ammonium moiety with Trp86 arises about equally from cation-pi and charge-independent interactions.