The existence of aminoglycoside-4′-nucleotidyltransferase [AAD(4′)] was demonstrated in the cell-free extract of B. brevis. This enzyme was purified about 100-fold over the cell-free extract by column chromatography using DEAE-cellulose and affinity chromatography using butirosin A-Sepharose 4B.AAD(4′) of B. brevis catalyzed the transfer of nucleotides to the 4′-hydroxyl group on amino-hexose, a constituent of 2-deoxystreptamine aminoglycosides. Ribo- and deoxyribonucleoside triphosphates were active as nucleotidyl donor. The pH optimum for the reaction was in the range of 5,5 to 6.0. AAD(4′) activity was dependent upon divalent metal-ions: Mn2+, Zn2+, Mg2+ and Co2+.The molecular weight as determined by gel filtration on a Sephadex G-100 column was approximately 45,000.