Isopenicillin-N synthetase (IPNS) was purified to homogeneity from Cephalosporium acremonium C0728. The enzyme existed in two states during purification; an oxidised state with a disulphide linkage and its reduced state. These two forms can be interconverted in the presence or absence of thiol agents, and separated by fast protein liquid chromatography (FPLC) with the strong anion exchange Mono-Q column. The enzyme is a monomer with a molecular mass of 38 kDa and pI 5.05. The first 50 amino acid N-terminal sequence of the enzyme was determined. The purified enzyme has an absolute requirement of Fe2+ and a 2-electron donor for activity.