The catalytic properties of the partially purified deacetoxycephalosporin C (DAOC)-synthetase and DAOC-hydroxylase from an industrial strain of Cephalosporium acremonium were studied. After mechanical breakage of the cells, purification was achieved by fractional (NH₄) ₂SO₄ precipitation, gel chromatography on Sephadex G-75, ion exchange chromatography on DEAE-Trisacryl M and two isoelectric focusing steps. The two enzyme activities could not be separated. Indirect evidence was obtained from SDS-polyacrylamide gel electrophoresis of the purest fractions obtained by isoelectric focusing that the two reactions are catalyzed by a single enzyme with a molecular weight of 33, 000±2, 000 and a pI of 4.6±0.1. Both reactions require α-ketoglutarate, FeSO₄, ascorbate and O₂, whereas additional ATP shows only a slight stimulation.