Staphylococcus aureus plasmids PSH2, RN1956 and pWA1 code for an aminoglycoside phosphotransferase; plasmid pWA1 also encodes an aminoglycoside-aminocyclitol adenylyltransferase. S. aureus plasmid pWA2 confers resistance to erythromycin and sulfonamide. Using plasmid ColE1-ApR (RSF2124) as a vehicle, we have transferred the genes determining aminoglycoside phosphotransferase and aminoglycoside-aminocyclitol adenylyltransferase activities from S. aureus to Escherichia coli. The new plasmids obtained confer aminoglycoside-aminocyclitol resistant phenotypes to E. coli, similar to, and by the same mechanisms as "naturally" occurring plasmids. By contrast, the results obtained after cloning of plasmid pWA2 indicate that certain S. aureus antibiotic resistance determinants (e.g. for erythromycin (Em) and sulfonamide (Su) cannot be phenotypically expressed in E. coli. The DNA of the constructed hybrid plasmids has been analysed by agarose gel electrophoresis following digestion with restriction endonucleases, by ultracentrifugation in cesium chloride, by hybridization, and by electron microscopy. Each hybrid is a cointegrate replicon, composed of an entire S. aureus plasmid covalently joined to ColE1-ApR.