4-Nitrophenyl-N-substituted carbamates (1–6) are the pseudo-substrate inhibitors of porcine pancreatic cholesterol esterase. Thus, the first step of the inhibition (K_i step) is the formation of the enzyme–inhibitor tetrahedral adduct and the second step of the inhibition (k_c) is the formation of the carbamyl enzyme. The formation of the enzyme–inhibitor tetrahedral adduct is further divided into two steps, the formation of the enzyme–inhibitor complex with the dissociation constant, K_S, at the first step and the formation of the enzyme–inhibitor tetrahedral adduct from the complex at the second step. The two-step mechanism for the formation of the enzyme–inhibitor tetrahedral adduct is confirmed by the pre-steady-state kinetics. The results of quantitative structure–activity relationships for the pre-steady-state inhibitions of cholesterol esterase by carbamates 1–6 indicate that values of −logK_S and logk₂/k₋₂ are correlated with the Taft substituent constant, σ*, and the ρ* values from these correlations are −0.33 and 0.1, respectively. The negative ρ* value for the −logK_S−σ*-correlation indicates that the first step of the two-step formation of the enzyme–inhibitor tetrahedral adduct (K_S step) is the formation of the positive enzyme–inhibitor complex. The positive ρ* value for the logk₂/k₋₂−σ*-correlation indicates that the enzyme–inhibitor tetrahedral adduct is more negative than the enzyme–inhibitor complex. Finally, the two-step mechanism for the formation of the enzyme–inhibitor tetrahedral adduct is proposed according to these results. Thus, the partially positive charge is developed at nitrogen of carbamates 1–6 in the enzyme–inhibitor complex probably due to the hydrogen bonding between the lone pair of nitrogen of carbamates 1–6 and the amide hydrogen of the oxyanion hole of the enzyme. The second step of the two-step formation of the enzyme–inhibitor tetrahedral adduct is the nucleophilic attack of the serine of the enzyme to the carbonyl group of carbamates 1–6 in the enzyme–inhibitor complex and develops the negative-charged oxygen in the adduct.