Tetrahedral intermediates of the alpha-chymotrypsin acylation by N-protected amide dipeptide substrates having different leaving groups were analyzed by means of the molecular mechanic method. A predominant role of the C-terminal interactions in the leaving group protonation and in the development of a kinetic specificity was determined. The kinetic specificity of serine proteases was shown to be controlled by the transformation of various forms of tetrahedral substrate intermediates at the presteady stage.