Blood cultures were negative in 88 (14%) of 620 cases of infective endocarditis (IE) documented in France during a 1-year nationwide survey. In 15 of these 88 cases, the causative microorganism was identified: seven cases of Q fever endocarditis and two cases of chlamydial endocarditis were diagnosed by serological and/or immunohistologic techniques, and a pathogen was cultured from five surgically removed valves and one arterial septic embolus. Forty-two (48%) of the 88 cases involved patients who had received antibiotics before the first blood sample was taken for culture. Mortality was lower in this group than among patients who had not previously received antibiotics (7% vs. 22%, P = .05). Comparison of blood culture-negative cases of IE with blood culture-positive cases revealed that the former tended to occur more often on prosthetic valves (32% vs. 22%, P = .16), were more often left-sided (97% vs. 83%, P = .0009), less often included extracardiac symptoms at presentation (52% vs. 63%, P = .06), and were more often surgically treated (53% vs. 34%, P = .001). Mortality was similar regardless of the results of blood culture (15% vs. 21%, P = .18). This study showed that more than 10% of all cases of IE in France are still associated with negative blood cultures and confirmed that a search for pathogens such as Coxiella burnetii and Chlamydia species is worthwhile in this situation.