We used broad-range PCR amplification and sequencing to detect and identify bacterial DNA in 156 valves of patients treated for infective endocarditis (IE). Bacterial DNA was found more frequently in patients who underwent valve replacement while on antibiotic treatment for IE (60%) than in patients who had completed antibiotic treatment for IE (37%; P = 0.02). We found specific bacterial DNA in valves removed from 11 of 30 patients who had completed antibiotic treatment for IE. Six had no histological evidence of IE. The presence of DNA was significantly correlated with the presence of histologic lesions (P = 0.001) and with the presence of bacteria detected by Gram staining (P < 0.001). Bartonella and streptococci were detected for much longer after antibiotic treatment by PCR than other species (P = 0.047 and 0.04, respectively), and coagulase-negative staphylococci were detected for much shorter periods (P = 0.02). The finding that bacterial DNA was more likely to be detected in valves of patients with active IE than in patients who had completed antibiotic treatment for IE shows that bacterial DNA is cleared slowly. There was no significant correlation between the duration of antibiotic therapy and the presence of bacterial DNA in valves. Since the persistence of bacterial DNA in valves does not necessarily indicate the persistence of viable bacteria, the detection of bacterial DNA in valves from IE patients should be interpreted with caution, in particular in those patients with a past history of treated IE.