BACKGROUND. Blood culture-negative endocarditis (BCNE) may account for up to 31% of all cases of endocarditis. METHODS. We used a prospective, multimodal strategy incorporating serological, molecular, and histopathological assays to investigate specimens from 819 patients suspected of having BCNE. RESULTS. Diagnosis of endocarditis was first ruled out for 60 patients. Among 759 patients with BCNE, a causative microorganism was identified in 62.7%, and a noninfective etiology in 2.5%. Blood was the most useful specimen, providing a diagnosis for 47.7% of patients by serological analysis (mainly Q fever and Bartonella infections). Broad-range polymerase chain reaction (PCR) of blood and Bartonella-specific Western blot methods diagnosed 7 additional cases. PCR of valvular biopsies identified 109 more etiologies, mostly streptococci, Tropheryma whipplei, Bartonella species, and fungi. Primer extension enrichment reaction and autoimmunohistochemistry identified a microorganism in 5 additional patients. No virus or Chlamydia species were detected. A noninfective cause of endocarditis, particularly neoplasic or autoimmune disease, was determined by histological analysis or by searching for antinuclear antibodies in 19 (2.5%) of the patients. Our diagnostic strategy proved useful and sensitive for BCNE workup. CONCLUSIONS. We highlight the major role of zoonotic agents and the underestimated role of noninfective diseases in BCNE. We propose serological analysis for Coxiella burnetii and Bartonella species, detection of antinuclear antibodies and rheumatoid factor as first-line tests, followed by specific PCR assays for T. whipplei, Bartonella species, and fungi in blood. Broad-spectrum 16S and 18S ribosomal RNA PCR may be performed on valvular biopsies, when available.