Culture-negative endocarditis (CNE) accounts for 2.5% to 48% of all cases of infectious endocarditis (IE). Prior or concurrent antibiotic treatment at the time blood cultures are taken accounts for 45% to 60% cases of CNE; the remainder are caused by slow-growing and fastidious organisms. Although limited in sensitivity because of potential contaminating bacterial DNA, detection of bacterial 16S ribosomal (r) DNA (from the 16S rRNA gene) is nevertheless more sensitive than culture. It is accomplished by using polymerase chain reaction (PCR) that targets highly conserved regions of the 16S rRNA gene. The identity of noncultivated infecting agents can then be determined by sequencing PCR products and comparing them with known 16S rDNA sequences from a wide range of bacteria. This has served to broaden the etiologic diagnosis of CNE. We review the benefits and limitations of PCR to diagnose IE and we propose advances that will be necessary to secure a place for PCR in guiding therapy.