The physicochemical characterization and proof of structure are reported for several new macrolide antibiotics related to tylosin which have been obtained by fermentation of mutant strains of Streptomyces fradiae. Tylosin is a 16-membered macrolide antibiotic which is produced commercially by strains of Streptomyces fradiae and is composed of a substituted lactone (tylonolide), an amino sugar (mycaminose) and two neutral sugars (mycinose, mycarose). Recently, a series of mutants of S. fradiae which are blocked in specific steps in the biosynthesis of tylosin has been described. From an analysis of these mutant strains in fermentation, cofermentation, in vitro enzymatic and in vivo bioconversion studies, it was proposed that tylosin is assembled via a preferred series of biosynthetic steps. This proposal is further supported by in vitro studies recently reported by OMURA and coworkers. Since the substrate specificities of certain enzymes involved in tylosin biosynthesis are not particularly stringent, blocked biosynthetic steps involving those enzymes were readily bypassed in particular mutant strains of S. fradiae. Consequently, some of the mutants produced biosynthetic intermediates to tylosin in high yield while others produced shunt metabolites, many of which possessed antimicrobial activity. Since many of these biosynthetic intermediates and shunt metabolites had not been previously reported, they were isolated and identified by physicochemical methods. After our work had been completed, a communication from another group was published describing a series of compounds which had been obtained from an independently-derived set of mutants which were blocked in various steps of tylosin biosynthesis. In this paper, we report our work on the isolation and elucidation of structure of the 16-membered macrolides which have been obtained from the biosynthetically-blocked mutants of S. fradiae previously described.