We recently described acidified citrate cysteine medium (ACCM), which supports host cell-free (axenic) growth of Coxiella burnetii. After 6 days of incubation, greater than 3 logs of growth was achieved with the avirulent Nine Mile phase II (NMII) strain. Here, we describe modified ACCM and culture conditions that support improved growth of C. burnetii and their use in genetic transformation and pathogen isolation from tissue samples. ACCM was modified by replacing fetal bovine serum with methyl-β-cyclodextrin to generate ACCM-2. Cultivation of NMII in ACCM-2 with moderate shaking and in 2.5% oxygen yielded 4 to 5 logs of growth over 7 days. Similar growth was achieved with the virulent Nine Mile phase I and G isolates of C. burnetii. Colonies that developed after 6 days of growth in ACCM-2 agarose were approximately 0.5 mm in diameter, roughly 5-fold larger than those formed in ACCM agarose. By electron microscopy, colonies consisted primarily of the C. burnetii small cell variant morphological form. NMII was successfully cultured in ACCM-2 when medium was inoculated with as little as 10 genome equivalents contained in tissue homogenates from infected SCID mice. A completely axenic C. burnetii genetic transformation system was developed using ACCM-2 that allowed isolation of transformants in about 2 1/2 weeks. Transformation experiments demonstrated clonal populations in colonies and a transformation frequency of approximately 5 × 10(-5). Cultivation in ACCM-2 will accelerate development of C. burnetii genetic tools and provide a sensitive means of primary isolation of the pathogen from Q fever patients.