Experiments at elevated temperatures permit the determination of rate constant and thermodynamic activation parameters for the neutral hydrolysis of the neurotransmitter acetylcholine in water. At 25 C, the extrapolated rate constant for the uncatalyzed (or neutral) hydrolysis of acetylcholine is 3.9 x 10(-7) s(-1) at 25 degrees C (Delta H(double dagger) = 20.0 kcal/mol; T Delta S(double dagger) = -6.1 kcal/mol). Acetylcholine is more susceptible to neutral and base-catalyzed hydrolysis than ethyl acetate but less susceptible to acid-catalyzed hydrolysis. For acetylcholinesterase from the electric eel, the catalytic proficiency [(k(cat)/K(m))/k(neutral)] is 2 x 10(16) M(-1), comparable in magnitude with the catalytic proficiencies of aminohydrolases that act on peptides and nucleosides.