During the course of a study of microbial transformation of macrolide antibiotics, we previously discovered 4"-Oacyltylosins with greater antimicrobial activity against macrolide-resistant Gram-positive bacteria and mycoplasmas than tylosin itself. Therefore, we investigated macrolides produced by mutants of a tylosin-producing strain, Streptomyces fradiae NRRL 2702. Spore suspensions of this strain were treated with N-methyl-N'-nitro-N-nitrosoguanidine, and about 12,000 colonies were examined, yielding 40 mutants that did not produce tylosin. Some of these mutants produced new macrolide compounds (I, II, IIIa, IIIb, IIIc, IIId, IVa, IVb, IVc, etc.). Compounds I and IVc were identified as protylonolide and macrocin, respectively, via 1H NMR, 13C NMR, and mass spectroscopy. The chemical structures of these new compounds were determined by spectroscopy (IR, 1H NMR, 13C NMR) and chemical degradation, showing they differ from tylosin in the mycinose moiety. Specifically, II was demycinosyl-23-deoxytylosin, IIIa (identical to IVb) was demycinosyltylosin, and IVa was demethylmacrocin. The structure of the new sugar moiety of IIIb was also proposed.