Production of sagamicin and gentamicin C1 in Micromonospora sagamiensis was regulated by cobalt ion. In a parental strain, KY11510, cobalt ion stimulated gentamicin C1 production and suppressed sagamicin production. By ultraviolet light or N-methyl-N'-nitro-N-nitrosoguanidine treatment, six mutants blocked in gentamicin C1 biosynthesis were obtained from KY11510. These mutants were classified into two types. The first type, four mutants, produced no gentamicin C1 even when cobalt ion was added to the fermentation. The second type, two mutants, produced a small amount of gentamicin C1 when a high concentration of cobalt ion was added. Based on biotransformation experiments, these mutants appeared to be blocked at the 6'-C-methylation step in the biosynthesis of gentamicin C1. The mutants showed an increased production of sagamicin. In addition, cobalt ion stimulated sagamicin production in the mutants. The mechanism of cobalt regulation in the parent and the mutants is discussed.