Q fever, caused by Coxiella burnetii, may be acute or chronic. Only a few strains of C. burnetii have been isolated due to the difficulty and hazard of isolation. We report here the isolation using a centrifugation shell vial technique of 16 new strains from patients suffering chronic Q fever. Twenty-four samples were inoculated onto human embryonic lung (HEL) fibroblast cell monolayers growing in shell vials. C. burnetii was detected 6 days later by using immunofluorescence. Samples from valves (n = 10), arterial prostheses (n = 2), bone (n = 3), skin biopsy (n = 1), bone marrow (n = 1), and blood (n = 5) from 16 patients were successfully cultured. Two cerebrospinal fluid samples from two patients were negative. The strains were subcultured in HEL cells and are now established. The technique is sensitive and less hazardous than animal inoculation. We recommend the shell vial technique for isolation of C. burnetii.