A novel series of milbemycin antibiotics were isolated from the fermentation broth of a Streptomyces species designated E225. The structures of the four main metabolites VM 44857 (1), VM 44864 (2), VM 44865 (3) and VM 44866 (4) were determined by NMR techniques. In addition we describe the solution conformations of the major metabolite VM 44857 (1).