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Cholinesterases (ChEs) display a non-michaelian behavior with positively charged substrates. In the steady-state rate equation, the b factor describes this behavior: if <i>b ></i> 1 there is substrate activation, if <i>b <</i> 1 there is substrate inhibition. The mechanistic significance of the <i>b</i> factor was investigated to determine whether this behavior depends on acylation, deacylation or on both steps. Kinetics of human acetyl- (AChE) and butyryl-cholinesterase (BChE) were performed under steady-state conditions and using a time-course of complete substrate hydrolysis. For the hydrolysis of short acyl(thio)esters, where acylation and deacylation are partly rate-limiting, steady-state kinetic analysis could not decide which step determines <i>b</i>. However, the study of the hydrolysis of an arylacylamide, 3-(acetamido)-N,N,N-trimethylanilinium (ATMA), where acetylation is rate-limiting, showed that <i>b</i> depends on the acylation step. The magnitude of <i>b</i> and opposite <i>b</i> values between AChE and BChE for the hydrolysis of acetyl(thio)- versus benzoyl-(thio) esters, then indicated that the productive adjustment of substrates in the active center at high concentration depends on motions of both the Ω and the acyl-binding loops. Benzoylcholine was shown to be a poor substrate of AChE, and steady-state kinetics showed a sudden inhibition at high concentration, likely due to the non-dissociation of hydrolysis products. The poor catalytic hydrolysis of this bulky ester by AChE illustrates the importance of the fine adjustment of substrate acyl moiety in the acyl-binding pocket. Molecular modeling and QM/MM simulations should definitively provide evidence for this statement.