Dansyl-Phe-Ala-Arg and (D-Ala2, Leu5) enkephalin-Arg6 (dalargine) were studied as possible substrates for enkephalin-forming carboxypeptidase (EFC) and carboxypeptidase B (CPB) in crude tissue homogenates. Activities of these enzymes in rat cerebellum, pituitary and adrenal glands were determined using the dansyl substrate. Kinetic characteristics of CPB and EFC were evaluated for dansyl substrate and dalargine. Affinity of EFC to dalargine was 10-fold higher as compared with CPB. Dalargine may be used as a specific substrate for study of EFC in tissue preparations.