By the use of improved isolation and separation methods, it has been shown that the major components of the nebramycin complex produced by Streptomyces tenebrarius are factor 2 (apramycin), factor 4 (6"-O-carbamoylkanamycin B), and factor 5' (6"-O-carbamoyltobramycin). Factor 5, which is identical to kanamycin B, and tobramycin are not produced by Streptomyces tenebrarius but arise from the acid or base catalyzed hydrolysis of 6"-O-carbamoylkanamycin B and 6"-O-carbamoyltobramycin respectively.