Penicillin acylase (EC 3.5.1.11) from E. coli, both in solution and immobilized on solid supports, has been commercially exploited for the large scale production of 6-aminopenicillanic acid (6-APA), which is an important intermediate for the manufacturing of semisynthetic penicillins. In this paper a very simple procedure of penicillin acylase purification is reported, which employs only one affinity chromatographic step (Sepharose-phenylacetic column). The enzyme was obtained at a high degree of purity and could be used for immobilization on partially hydrolyzed and activated nylon. Since the support is chemically inert and mechanically stable the catalyst can be used several times without any significant loss of activity, making the process of great commercial importance.