The genes rdmB and rdmC of Streptomyces purpurascens encoding aclacinomycin modifying enzymes RdmB and RdmC were expressed in Streptomyces lividans TK24. In contrast to the earlier suggestion that RdmC may be an esterase that causes the removal of the carbomethoxy group from the 10 position of aclacinomycins, RdmC functions as an aclacinomycin methyl esterase and catalyzes the removal of the methoxy group from the C-15 position of aclacinomycin T producing 15-demethoxyaclacinomycin T. RdmB acts upon C-10 of 15-demethoxyaclacinomycin T and is able to remove the carboxylic group from the C-10 position. It functions also as an aclacinomycin-10-hydroxylase being able to add a hydroxyl group at the same, C-10 position in vitro. Aclacinomycin methyl esterase was purified to apparent homogeneity from S. lividans carrying the rdmC and aclacinomycin-10-hydroxylase as a glutathione S-transferase fusion construct from Escherichia coli carrying the rdmB gene, respectively. Aclacinomycin methyl esterase functions as a monomer and aclacinomycin-10-hydroxylase as a tetramer. Aclacinomycin methyl esterase has an exceptionally high temperature stability and has an apparent K(m) for aclacinomycin T of 15.5 microM. The introduction of rdmC and rdmB in a Streptomyces galilaeus mutant HO38 produced the same modifications of aclacinomycin T in vivo as aclacinomycin methyl esterase and aclacinomycin-10-hydroxylase in vitro.