Dipeptidyl-aminopeptidase I (dipeptidyl-peptide hydrolase, EC 3.4.14.1) from bovine spleen has been immobilized by hydrophobic bonding to alkyl- or aryl-Sepharoses. Optimum binding occurred with octyl- and phenyl-Sepharoses. The activity of the immobilised dipeptidyl-aminopeptidase I has been determined using glycylarginyl-p-nitroanilide as substrate and the pH optimum of the immobilised enzyme determined as well as the stability of the enzyme to repeated use. Preliminary studies using immobilised dipeptidyl-aminopeptidase I for the digestion of methionine enkephalin have been carried out using reverse-phase HPLC to analyse the reaction.