In a neomycin-producing strain Streptomyces fradiae, aminoglycoside-inactivating activities (including Aph(3')-V) contribute to antibiotic resistance. Butirosin A is a poor substrate for Aph(3')-I and V. To investigate the resistance mechanism, butirosin A-resistant mutants were isolated from S. fradiae IFO 3718. These mutants were categorized into three groups based on their antibiotic susceptibility profiles and ability to inactivate xylostasin and butirosin A. The third group (BSR-15 and BSR-52) showed unique resistance patterns and produced unknown inactivation products from xylostasin and butirosin A. Detailed chemical analysis of the inactivated butirosin A from BSR-15 using IR, ¹H-NMR, and ¹³C-NMR spectrometry revealed that it was 2'-N-acetylbutirosin A, indicating that butirosin A was inactivated by 2'-N-acetylation in these mutants.