Bartonella endocarditis is a severe disease for which blood cultures frequently remain negative. We tested three PCR assays by using specimens of serum sampled early during the disease from 43 patients diagnosed in our laboratory as having Bartonella endocarditis on the basis of serological, culture, and/or valvular molecular detection. We tested a two-step nested PCR (TSN-PCR), a one-step nested PCR (OSN-PCR) with a regular thermal cycler, and a one-step nested PCR with the LightCycler (LCN-PCR). These assays were performed with primers derived from the riboflavin synthase-encoding gene ribC, never before amplified in our laboratory. Due to contamination of negative controls, the results of the TSN-PCR were not interpretable, and this technique was no longer considered. The LCN-PCR had a specificity of 100% and a sensitivity of 58.1%, higher than those of the OSN-PCR (18.6%; P < 0.01) and prolonged blood culturing (7.1%; P < 0.01). The LCN-PCR results correlated strictly with those of other direct diagnostic tests, when available, and identified the causative species for six patients previously diagnosed on the basis of serological analysis only. The efficacy of the LCN-PCR was not influenced by antibiotics (P = 0.96) but was altered by prolonged storage of serum specimens at -20 degrees C (P = 0.04). Overall, the LCN-PCR is specific and more sensitive than traditional methods (i.e., culturing and/or PCR with EDTA-treated blood). It can easily be applied to the diagnosis of patients with suspected Bartonella endocarditis, especially when only serum is available.