A disadvantage in Q fever diagnostics and research is the insensitive and difficult culture of Coxiella burnetii. This intracellular organism can only be isolated using embryonated eggs, animal hosts, or mammalian cell culture. In consequence, it has only been possible to isolate a few strains from human patients. Here, we describe the first isolation of C. burnetii from a clinical specimen using the recently developed cell-free medium acidified citrate cysteine medium 2 (ACCM2). We screened the sera of 217 patients who had undergone valvular transplantation but detected one serum with an antibody constellation indicating chronic Q fever. Polymerase chain reaction (PCR) of the corresponding heart valve revealed 3.1 × 10(5) copies/rxn. The strain was successfully isolated using ACCM2. Genomic investigation by multilocus variable-number of tandem repeat (VNTR) analysis (MLVA) revealed the strain to be a new genotype, A10, closely related to one from sheep. As the sensitivity of ACCM2 for different human strains is unknown, we also investigated combining a robust test, egg propagation, with ACCM2. This combination produced four to six logs of growth of the bacteria. The use of ACCM2 in this combination simplified the otherwise elaborate purification steps. Cultivation in ACCM2 has the potential to simplify the isolation of C. burnetii in a clinical setting. As the success rates of cell culture for virulent C. burnetii strains are variable, the sensitivity of ACCM2 for different strains is unknown, and many specimens may contain much fewer bacteria than in our case, the combination of the robust method of egg propagation with ACCM2 is a good alternative to existing single methods for investigating critical specimens.