Mechanisms of phase variation in Coxiella burnetii, the etiological agent of Q-fever, were studied using variously virulent strains with different lipopolysacharide structures to obtain possible molecular correlates of attentuation. A. Genetic heterogeneity. Chromosomal DNA from the Nine Mile phase I strain of C. burnetii (CB9MIC7) cloned into the cosmid vector pHC79 was used as a probe to show a HAE III fragment present in the parent strain but absent from a spontaneously derived Nine Mile phase II strain (CB9MIIC4). An 18 kb deletion in the chromosomal DNA of CB9MIIC4 was identified. Another intrastrain spontaneous derivative, CB9M1514, also lacked the sentinel Hae III fragment. This strain carried a deletion which was approximately 29 kb, and both deletions appeared to share a common terminus within the limits of resolution. In all other strains investigated, both phase I and phase II, the DNA represented by the insert seemed grossly intact. B. Lipopolysaccharide. LPSs extracted from nine strains of C. burnetii were analyzed for chemical compositions, molecular heterogeneity by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and lethal toxicities in galatosamine sensitized mice. The structure of a unique disaccharide prepared from the CB9MIC7 strain was determined using negative ion extraction fast atom bombardment mass spectrometry of the N-acetylated disaccharide, direct chemical ionization mass spectrometry of the N-acetylated then permethylated disaccharide, and DCI and FAB mass spectrometry of reduced permethylated and peracetylated derivatives. The chemical structure was described as galactosaminuronyl-yield(1-6)-glucosamine [GalNU-yield(1-6)-GlcN], (C12H22N2)10] and Mr of 354. Using GalnU-yield(1-6)-G1cN and two recently described sugars, virenose and dihydrohydroxystreptose, as biochemical markers of truncated LPSs, we were able to relate Mr of selected molecular species of LPSs to truncation of LSP of C. burnetii intra- and inter- specific strains. Smooth-type LPS contained all three compounds, semi rough LPS did not contain virenose, and rough LPS was deficient in all three components. All of the LPSs were toxic in galactosamine sensitized mice albeit they were 100- to 1000-fold less toxic than Escherichia coli and Salmonella typhimurium endotoxins. Significance: Identification of chromosomal and plasmid DNA participating in virulence expression and LPS biosynthesis will facilitate our understanding of phase variation and endotoxin activities of C. burnettii.