Enediyne antibiotics are known for their potent anti tumor activities. Neocarzinostatin (NCS) is produces by Streptomyces carzinostaticus and comprised 1:1 complex of non-peptide chromophore and peptide apoperotein (NcsA). Adapting a strategy for cloning NCS biosynthetic genes, the naphthoate synthase (NS) gene was localized in a cosmid that expanded 33.0 kb. One of the components of NCS, naphthoic acid moiety is produced via PKS pathway by the six intact acetate units. The 5.45 kb iterative type I PKS, NS responsible for its biosynthesis shares sequence homology with 6-MSAS of fungi origin as well as with AviM and CalO5 of Streptomyces origins. It was heterologously expressed in Streptomyces hosts and the major isolated product identified was 2-hydroxy-5-methyl-1-naphthoic acid. This result shows the involvement of this NS gene in NCS biosynthesis. To date iterative type I PKS for monocyclic aromatic compound have been found in limited Streptomyces. This is the first report of similar bacterial PKS from enediyne producer that enables the biosynthesis of bicyclic aromatic compound.