Secondary metabolites including polyketides, nonribosomal peptides and hybrid polyketide-peptides have diverse chemical structures and a wide variety of bioactivities.1 Polyketides and nonribosomal peptides are synthesized by large enzyme complexes such as polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS).2 These biosynthetic proteins are typically encoded in neighboring loci and organized in gene clusters ranging from several to over 200 kb in length. NRPS and PKS employ a very similar strategy like an assembly line for the biosynthesis of two distinct classes of natural products.3–5 In addition, NRPS/PKS hybrid biosynthetic systems give structurally more diverse compounds by the combination of NRPS and PKS like building blocks on one assembly line.6The biosynthetic gene cluster of a polyketide kirromycin7 (3, Figure 1b) was identified from the genome sequence of Streptomyces collinus8–10 and kirromycin was indicated to be biosynthesized by a large hybrid PKS/NRPS gene cluster. Among the related compounds including efrotomycin,11 dihydromocimycin,12 heneicomycin,13 factumycin14 and kirromycin,15 only the biosynthesis of kirromycin has been reported so far.8–10 Previously, a new kirromycin analog L-681,217 (2, Figure 1a), which lacked pyridine ring was isolated from Streptomyces cattleya ATCC 39203.16 On the basis of these results, we performed chemical investigation on a MeOH extract of S. cattelya NBRC 14057 (type strain) to search for new kirromycin analogs. As a result, a new kirromycin analog demethyl-L-681,217 (1) was isolated as an antibacterial compound from S. cattelya along with a known compound L-681,217 (2). As the genome sequence of the type strain of S. cattletya was previously determined,17 the biosynthetic gene cluster was searched for and found based on the similarity to that of kirromycin. Here we describe the isolation and structure determination of a new kirromycin analog demethyl-L-681,217 (1) and discuss the possible biosynthetic gene cluster.