Abstract Clorobiocin (18631 RP) has been hydrolysed at the amide link to give the 3-aminocoumarin, clorobiocinamine, using an enzyme from the bacterium NRRL B-3652. An assay was developed which depended on the appearance of the ultraviolet absorbance of clorobiocinamine at 345 nm. The enzyme was partially purified by sonication, centrifugation, and ammonium sulphate fractionation. The enzyme activity had a pH optimum between 7.0 and 8.0. Clorobiocin inhibited the enzyme at concentrations above 20 μ m probably because clorobiocinamine inhibited the enzyme irreversibly. In order to prepare clorobiocinamine in quantities of the order of 0.1 g, the reaction was carried out in an Amicon 402 ultrafiltration cell using clorobiocin (0.1%) and a high enzyme level (10 000 units). Under these conditions the reaction went to completion. Clorobiocinamine was collected and purified, ready for use in chemical syntheses of clorobiocin analogues.