The nucleotide (nt) sequence of a 3409-bp PvuII fragment carrying the self-defense gene (fmrO) of a fortimicin A (FmA; astromicin) producer, Micromonospora olivasterospora, was determined; four open reading frames (ORFs) exist in this region. Expression analysis of fmrO using the lac promoter in Escherichia coli revealed that ORF-3 encodes fmrO. ORF-1 was considered to encode a FmKH epimerase (fms11). ORF-2, ORF-3 and ORF-4 seemed to form an operon. No homology was detected between fmrO and the resistance-encoding gene fmrT of Streptomyces tenjimariensis, a producer of the Fm-group antibiotic, istamycin. Gene organization around the fmr genes differed considerably between M. olivasterospora and S. tenjimariensis. The deduced amino acid sequence of fmrO showed an identity of 30.8% to grmA of M. purpurea and 35.8% to grmB of M. rosea, respectively, suggesting that fmrO encodes a 16S rRNA methyltransferase. We found in M. purpurea a novel resistance gene distinct from grmA and highly similar to fmrO which conferred a resistance phenotype similar to that of fmrO. These results suggest that fmrO of M. olivasterospora and the two resistance-encoding genes of M. purpurea were derived from a common ancestral gene.