Staphylococcus aureus and coagulase-negative staphylococcal isolates from various geographic areas in the United States were examined by using a conjugative transfer gene DNA probe in dot-blot hybridization assays. Of 175 S. aureus isolates, 47 (27%) hybridized with the probe, while 24 of 208 (11.5%) coagulase-negative staphylococci hybridized. However, among methicillin-resistant S. aureus 52% (45 of 89) were probe positive while only 2% (2 of 86) of methicillin-susceptible S. aureus were probe positive. In contrast, 12.5% (22 of 176) of methicillin-resistant and 6% (2 of 32) of methicillin-susceptible coagulase-negative staphylococci contained transfer genes. All but one of the staphylococci containing transfer genes were resistant to gentamicin; 91.5% of S. aureus and 65% of coagulase-negative staphylococci containing transfer genes transferred gentamicin resistance to a S. aureus recipient. Of the 12 isolates that hybridized with the probe but did not transfer resistance, 10 (6 coagulase-negative staphylococci and 4 S. aureus) carried both gentamicin resistance and conjugative transfer genes on the same plasmid. Of these 10, 6 contained plasmid target fragments of sizes different from that of the probe, suggesting additions or deletions of DNA essential for transfer, while in 4 no such alterations could be detected. In two coagulase-negative staphylococci the entire transfer region was apparently integrated into the chromosome. Thus, staphylococci carrying conjugative transfer genes are widely disseminated in the United States and are usually found in multiresistant isolates on plasmids that also encode gentamicin resistance.