We used the polymerase chain reaction (PCR) and broad-range bacterial primers, combined with DNA sequencing, to identify Bartonella quintana as the etiologic agent in a case of culture-negative infective endocarditis; all blood cultures, as well as the bacterial cultures of the resected aortic valve and vegetations, remained negative. PCR was used to amplify bacterial 16S rDNA from a template prepared from the aortic valve vegetation. The amplified 16S rDNA produced a nucleotide sequence that was 99.79% identical to the B. quintana rDNA sequence. The patient had a highly elevated level of serum antibodies to Bartonella antigen (1:8,192).