13C Labelled α-ketoglutarates in conjunction with 18O2 were used to investigate the role of α-ketoglutarate in cephalosporin biosynthesis. [2-13C]- and [1,2-13C]-α-ketoglutarates were synthesised and incubated with recombinant DAOC/DAC synthetase. 13C NMR revealed α-ketoglutarate was converted to [1-13C]succinate. Incubations under 18O2 showed >90% 18O incorporation into succinate but only ca. 50% into deacetylcephalosporin C (DAC). Stoichiometry of β-lactam substrate:α-ketoglutarate was 1:1 for both penicillin N→deacetoxycephalosporin C (DAOC) and DAOC→DAC conversions. These results are consistent with a mechanism involving an iron 'oxene' moiety generated by enzyme-bound Fe²+ with dioxygen and α-ketoglutarate, and the less than complete 18O incorporation into DAC is probably due to oxygen exchange with water before substrate oxidation.