Streptomyces sp. NR0489 produces tetronothiodin, a novel brain-type cholecystokinin receptor antagonist. This species was differentiated from its related species S. gelaticus, S. griseolus and S. hydrogenans on the basis of their cultural characteristics, such as the utilization of carbohydrates and the presence or absence of various enzymatic activities. We applied the DNA-DNA hybridization method using photobiotin, which proved the genetic difference between the four species mentioned above. The yield improvement effort including single colony isolation, mutation, and protoplast regeneration together with medium optimization resulted in more than an 81-fold increase of the productivity of tetronothiodin as compared to that of the wild type strain.