Inhibition of penicillin acylases from Escherichia coli and Alcaligenes faecalis by aliphatic and aromatic alcohols was studied. It was shown that the inhibition of both enzymes has competitive nature and they bind the alcohols at the acyl group binding site of the enzyme active center. The free energy of alcohol sorption was shown to be linearly dependent on the hydrophobicity of the inhibitor with slopes of 1.6 and 1.7, demonstrating extremely effective hydrophobic interactions. To rationalize the observed distinctions in the inhibiting properties of aromatic and aliphatic alcohols beginning with butanol, it was suggested that the loss of entropy occurring on the interaction of the ligand with the tightly restricted hydrophobic pocket of the active center makes an essential contribution to the overall energetics of complex formation.