Resistance (R) factor-mediated streptomycin (Sm) resistance differs from classical, high-level, chromosome-borne Sm resistance in its dominance over sensitivity and in the level of its effectiveness (in Escherichia coli approximately 25 mug/ml versus >2,000 mug/ml). In addition, an R factor-containing strain, unlike high-level Sm-resistant bacteria, showed an inoculum effect with respect to its level of Sm resistance. Crude extracts of this strain destroyed the inhibitory activity of Sm and bluensomycin (Blue) on in vitro protein synthesis. The ribosomes from this strain proved to be sensitive to Sm in vitro. The requirements for in vitro inactivation of Sm (and Blue) were determined to be: extract, adenosine triphosphate or deoxyadenosinetriphosphate, and Mg(++). Chromatographic techniques with radioisotopes revealed the formation of an inactivated form of Sm containing adenosine (or deoxyadenosine), phosphate, and Sm in equimolar amounts. The adenylate moiety is coupled to the streptobiosamine residue, rather than to the streptidine ring, of the Sm molecule. The adenylating enzyme, which is not induced by Sm, is located in the periplasmic space of the R factor-containing strain.