The gene for isopenicillin N synthase (cyclase; IPNS) was cloned from Lysobacter lactamgenus using DNA probe amplified with primers based on the consensus sequences of isopenicillin N synthase genes of other ${eta}$-lactam-producing microorganisms. The genomic library of L. lactamgenus using pUC18 plasmid cloned at the SacI site were screened with the PCR-generated DNA probe and three positive clones were isolated. Enzyme activities in E. coli clones were confirmed by bioassay and HPLC assay. Throughout the functional mapping, it was observed that the gene for isopenicillin N synthase is located at the 1.3-kb XhoI-BamHI fragment of insert of positive clones. Nucleotide sequencing at both ends of the XhoI-BamHI fragment revealed that IPNS of L. lactamgenus has the common amino acid sequences at amino- and carboxy-termini.