Increasing resistance to aminoglycoside antimicrobial agents in clinical isolates of Staphylococcus aureus was encountered in one hospital over a three-year period. Eleven of 16 isolates, selected to represent different time periods, transferred gentamicin resistance on filter membranes with transfer properties and restriction-enzyme analyses that were similar to pAM899-1, a self-transferable gentamicin plasmid previously isolated from Staphylococcus epidermidis within the same hospital. Self-transferable gentamicin R plasmids were demonstrated in isolates from six other nosocomial environments as early as 1974 and, though showing different restriction-enzyme digests, were highly related to pAM899-1 on the basis of Southern hybridization. These recently recognized, self-transferable plasmids seem to share a common evolutionary background and are important contributors to the increased resistance encountered in nosocomial staphylococci.