Site-directed mutagenesis studies have shown that the isopenicillin N synthase of Cephalosporium acremonium (cIPNS) requires two essential histidine residues (H216, H272) for activity. The determination of iron bound to the wildtype cIPNS and its absence in the mutants lacking histidine at positions 216 and 272 clearly supports the essential role these two histidines play in iron binding. However, nuclear magnetic resonance (NMR) studies have indicated that there could be three histidine residues that possibly coordinate the essential iron at the active site. To search for a presumed third histidine ligand, mutant cIPNS genes containing mutations at two histidine codons were created by in vitro cloning of fragments from the expression vectors bearing the respective cIPNS genes each with a single histidine mutation at positions H49, H64, H116, H126 and H137. All ten possible double histidine mutant cIPNS constructs were subsequently expressed in Escherichia coli. If a third histidine had a participatory role in the iron active centre of cIPNS, then one of the constructed double histidine mutants would have lost its enzymatic activity. However, analysis of the cIPNS activities of these recombinant double histidine mutants indicated that none of them was totally inactivated. Thus, the involvement of a third histidine can be repudiated.