1) The specificity of proteinase K towards amino acid and oligopeptide nitroanilide substrates is investigated. 2) The active center of the enzyme contains an extended binding region consisting of several subsites. An integral part of the S1-subsite are hydrophobic areas which were investigated by systematic elongation of the carbon skeleton in carboxylic acid 4-nitrophenyl esters. On the basis of these studies, a possible model of the S1-binding site is proposed. 3) Kinetic parameters for the hydrolysis of substituted phenyl acetates catalyzed by proteinase K have been measured at pH 7 and 25 degrees C. Deacylation of an acyl-enzyme intermediate is probably the rate-limiting step. Acylation (kcat/km used as a measure) is modestly sensitive to the sigma values of the substituents (p = 1.33, r = 0.9108), indicating electrophilic assistance by the enzyme in the catalytic mechanism. 4) Hydrophobic forces apparently are not involved in the binding of the leaving group.