Strains of Escherichia coli carrying R-factor R71(a), which codes for a streptomycin-spectinomycin adenylyltransferase, have elevated levels of resistance to dihydrostreptomycin (DHS) compared with isogenic R− bacteria. DHS accumulated by whole cells and spheroplasts of R+ bacteria is lower than that observed for R− strains, a result of the absence of the second and more rapid of the two energy-dependent phases of DHS uptake seen in susceptible E. coli. A mutant of R+E. coli with reduced DHS resistance has been shown to have reduced levels of streptomycin-spectinomycin adenylyltransferase activity as well as enhanced drug accumulation. Actively accumulated DHS was recovered from R+ cells as the adenylylated derivative. Neither was inactivated antibiotic detected in culture filtrates, nor was actively accumulated drug lost from R+ cells under normal conditions. The cellular distribution of actively accumulated DHS in R+ and R− cells was found to be the same. Membranes isolated from these cells retained only a small fraction (≃1%) of the total cell-associated drug. The R+ derivative of a mutant with defective energy transduction (E. coli NR-70) and reduced ability to transport aminoglycosides has a significantly higher minimal inhibitory concentration of DHS than its R+ parent (strain 7). Streptomycin-spectinomycin adenylyltransferase activity, from comparisons of Km values and total activities of enzyme, was the same in both strains. The enzyme has been localized to the exterior surface of the bacterial inner membrane, although isolated membranes lacked detectable enzyme activity. The preceding observations are consistent with the proposal that the level of R71(a)-mediated DHS resistance is the outcome of competition between the rate of adenylylation and the rate of the first energy-dependent phase of DHS transport. When the rate of adenylylation exceeds the first energy-dependent phase, adenylylated DHS is accumulated, apparently in a manner identical to the accumulation of DHS. Unlike DHS, adenylylated DHS does not interact with ribosomes, and, consequently, there is a failure to initiate ribosomally dependent sequelae such as the second energy-dependent phase of accumulation, inhibition of protein synthesis, and/or misreading of mRNA.