The isopenicillin N synthase of Cephalosporium acremonium (cIPNS) involves a catalytically important non-heme iron which is coordinated credibly to histidine residues. A comparison of the IPNS genes from various microbial sources indicated that there are seven conserved histidine residues. These were individually replaced by leucine residues through site-directed mutagenesis, and the sites of mutation were confirmed by DNA sequencing. The seven mutant genes were cloned separately into the vector pET24d for expression in Escherichia coli BL21 (DE3), and the proteins were expressed as soluble enzymes. All the resulting mutant enzymes obtained have mobilities of approximately 38 kDa, identical with the wild-type enzyme on SDS-polyacrylamide gel electrophoresis, and were also reactive to cIPNS antibodies. The enzymes were purified by ammonium sulfate precipitation and DEAE-Sephadex A-50 ion exchange chromatography, and these were analyzed for enzyme activity. A group of mutant enzymes, H49L, H64L, H116L, H126L, and H137L, were found to be enzymatically active with reduced activities of 16-93.7%, indicating that they are not essential for catalysis. Two of the mutant enzymes, H216L and H272L, were found to have lost their enzymatic activity completely, indicating that both His-216 and His-272 are crucial for catalysis. It is suggested that these histidines are likely to serve as ligands for binding to the non-heme iron in the IPNS active site. Alignment of the amino acid sequence of IPNS to related non-heme Fe(2+)-requiring enzymes indicated that the two essential histidine residues correspond to two invariant residues located in highly homologous regions. The conservation of the two closely located histidine residues indicates the possible conservation of similar iron-binding sites in these enzymes.