A simplified enrichment method for selective isolation of Actinomadura rugatobispora strains from soil is described. A. rugatobispora strains poorly produced spores on agar media when 5 to 20 colonies grew on a plate while characteristic spore formation was observed on humic acid-MOPS gellan gum medium. Spores of A. rugatobispora strains were tolerant to dry heating treatment at 120°C for 60 min. A. rugatobispora strains were resistant to 50 μg/ml trimethoprim and 10 μg/ml nalidixic acid, both of which inhibited the growth of nonfilamentous soil bacteria. In addition, these strains were more resistant to 100 μg/ml gentamicin and 20 μg/ml ampicillin than Streptomyces strains, which interfere with the growth of A. rugatobispora selectively. Owing these characteristics, we were successful to isolate 260 strains of this species from 148 out of 547 soil samples (27% of the samples tested). They were recovered from the soil samples with pH values ranging from 5.0 to 8.6, and 91% of the strains were isolated from neutral to slightly alkaline soil (pH 6.0 to 9.0). These results indicated that A. rugatobispora strains were distributed worldwide.