A total of 517 strains of Staphylococcus aureus isolated at a hospital in Melbourne, Australia between 1946 and 1981 was examined for resistance to a range of antimicrobial agents and for the presence of plasmid DNA. The use of mixed-culture transfer and restriction endonuclease analysis showed that the determinants for resistance to penicillin and to the heavy metals were carried by several related plasmids of (15-23) X 10(6) mol. wt, and that tetracycline resistance was encoded on a plasmid of 2.8 X 10(6) mol. wt in strains isolated before 1970. These phenotypes were chromosomally encoded in the majority of strains isolated thereafter. Resistance to chloramphenicol throughout the study period was plasmid-mediated. Of five aminoglycoside-resistance phenotypes, one was plasmid-mediated and three were chromosomally encoded. The remaining phenotype, specifying low-level gentamicin resistance, was found to be located on the chromosome of early isolates, but in later strains was borne by an 18 X 10(6) mol. wt plasmid which also encoded resistance to quaternary ammonium compounds.