The enediyne antitumor antibiotic neocarzinostatin (NCS) is produced by Streptomyces carzinostaticus ATCC15944. The biosynthetic pathway for the naphthoic acid moiety (boxed) of the NCS chromophore (1) is proposed to comprise five enzymes: NcsB, NcsB1, NcsB2, NcsB3, NcsB4. Both in vivo and in vitro experiments were used to support the proposed pathway. NcsB2 was characterized for the ability to catalyze the activation of 2-hydroxy-7-methoxy-5-methyl-1-naphthoic acid (3) via a naphthoyl-AMP ester (5) and subsequent formation of the corresponding coenzyme A ester. The finding that NcsB2 exhibits promiscuous substrate specificity toward a diverse set of naphthoic acid analogues with 2-, 5-, or 7-substitutions presents an outstanding opportunity to produce novel analogues of 1 by engineering NCS biosynthesis.