Pulsed field gradient gel electrophoresis (PFGE) provides a powerful technique for the analysis of bacterial genomic DNA by allowing the resolution of DNA fragments as large as 9000 kilobase pairs (kbp). When isolates of Coxiella burnetii were examined using this method, the restriction enzymes Not I and Sfi I gave the fewest and most easily resolved fragments. Sfi I cuts the genome of the Priscilla isolate of C. burnetii into 15 DNA fragments ranging in size from 320 to 18 kbp, and Not I cuts the DNA of this isolate into 20 fragments from 293 to 10 kbp in size. Analysis of the undigested DNA and summing of the Sfi I restriction fragments both indicate that the C. burnetii DNA contains approximately 1600 kbp, or is about one-third the size of the DNA in Escherichia coli. Comparisons of isolates revealed that the numbers and patterns of DNA fragments observed correlate with proposed strain designations. Because PFGE allows the reproducible separation of restriction endonuclease-digested C. burnetii DNA fragments into precise bands, it greatly facilitates the selection of large DNA fragments for cloning. Bands harvested from the gel can be cloned. Clone banks are invaluable for identifying the location of specific genes and landmarks and providing material for future experiments, including DNA sequencing. Yeast artificial chromosome (YAC) cloning vectors can accept fragments as large as 500 kbp. The fragmentation patterns of C. burnetii that we have obtained with infrequent-cutting enzymes are small enough to be cloned into YAC vectors. Using a PFGE selection method means that only small libraries would have to be created and screened. Thus, the results of these experiments also demonstrate the applicability of PFGE for deriving a physical map of C. burnetii chromosomal DNA. Development of such a macrorestriction map will facilitate genetic and molecular studies with C. burnetii.