The isolation and structure determination of 3-N-acetylribostamycin, a microbiologically inactive derivative, produced enzymatically from ribostamycin by Streptomyces ribosidificus is described. The location of the acetyl group was established by mass and NMR spectrometry of the new compound and its derivatives, and by optical rotation studies conducted on N-ethoxycarbonyl-2-deoxystreptamine. The latter compound was obtained by partial acid hydrolysis of tri-N-ethoxycarbonyl-N-acetylribostamycin.