The obligate intracellular bacterium Coxiella burnetii is the etiological agent of acute Q-fever and chronic endocarditis in humans and of several zoonotic infections. The DNA from a variety of these disease isolates was compared for homology to the plasmid QpH1, found in the Nine Mile strain. Three patterns of homology were found in these isolates, i.e., one pattern identical to that of QpH1, one common to several endocarditis isolates and goat abortion isolates, and one common to the remaining group of endocarditis isolates. Plasmid DNA from the endocarditis-abortion isolate group, designated QpRS, was mapped by restriction enzyme analysis and compared with QpH1. These data show that QpRS was 2 to 3 kilobase pairs larger, contained DNA not found in QpH1, but was not generated from QpH1 by a single insertional event. Isolation of plasmid DNA from the second endocarditis group of isolates was not successful and may indicate that the plasmid has integrated into the chromosome. This analysis provides the first clear evidence that differences exist between C. burnetii isolates which cause various diseases, indicating that different C. burnetii strains may have unique virulence characteristics.