A key step which involves the cyclization of &dgr;-(L-&agr;-aminoadipyl)-L-cysteinyl-D-valine to the bicyclic ring structure of isopenicillin N in the penicillin and cephalosporin biosynthetic pathway, is catalyzed by isopenicillin N synthase (IPNS). In this study, an IPNS gene fromStreptomyces lipmaniiNRRL 3584 (slIPNS) was cloned via PCR-based homology cloning, sequenced and expressed inEscherichia coli. Soluble slIPNS was overexpressed up to 21% of total soluble protein, and verified to be functionally active when in an IPNS enzymatic assay. Sequence comparison of the slIPNS gene obtained (excluding the consensus primer sequences) with another cloned IPNS fromS. lipmanii16884.3, revealed one three-nucleotide deletion and three closely-spaced single nucleotide deletions. Futhermore, this paper also reports the first instance of the usage of PCR as an alternative and rapid strategy for IPNS cloning using consensus primers.Key words: isopenicillin N synthase, &bgr;-lactam antibiotics, secondary metabolism, consensus primers.