The etiology of blood culture-negative endocarditis was identified in 62.7% of 759 patients using combinations of serological, molecular, and histopathological assays. The majority of the agents not detected by conventional cultures were Coxiella burnetii (30%) and Bartonella spp. (11%), both diagnosed using serological tests, and Trophyrema whipplei, other unusual bacteria, and fungi making up the remainder. For 14% of patients, diagnosis required PCR testing on valve tissue removed during surgery (not in paraffin). The cause of disease could not be determined for 264 patients; 2% of patients had non-infectious endocarditis.