The polyene macrolides nystatin A1 and amphotericin B are effective but toxic antifungal antibiotics that are also active against enveloped viruses, protozoan parasites and pathogenic prion proteins. This chapter describes methods for genetic manipulation of the amphotericin and nystatin producers, Streptomyces nodosus and Streptomyces noursei. These techniques have been used to engineer the biosynthesis of several analogues of both polyenes. Methods for production, identification, purification and characterization of new analogues are also discussed.