N,N'-Dicyclohexylcarbodiimide (DCCD) was found to inhibit the activity of proline-beta-naphthylamidase purified from porcine intestinal mucosa. The inhibition is rapid and reversible, and it is not due to the dissociation of the enzyme subunits. The mode of the inhibition by DCCD is noncompetitive with respect to each of the two substrates tested. Ki values of DCCD for the enzyme were determined to be 1.9 microM with proline-beta-naphthylamide and 12 microM for L-leucine ethyl ester. To our knowledge, this is the first time that DCCD was found to be a potent, reversible inhibitor for an enzyme.