A new inactivated product of ribostamycin (SF-733), 3-N-carboxymethyl ribostamycin, was obtained from the broth of Streptomyces ribosidificus which was grown on a medium containing D-xylose. Detection and some biochemical mechanism of N-carboxymethylation were discussed, and structure of 3-N-carboxymethyl ribostamycin was proposed based on the chemical degradation and synthesis.