The molecular approach of PCR amplification of specific gene targets and universal loci for bacteria (16S rRNA) and fungi (18S, 28S and 5.8S rRNA) and subsequent sequencing was used to identify the possible causal microbial agent(s) in blood culture (47 patients) and heart valve material (30 patients) from patients with suspected infective endocarditis (IE). Culture and molecular results were analysed with respect to the patients' clinical background and the Duke Criteria. The findings demonstrated that: (i) all patients who were definite or possible cases were positive by PCR, even patients whose blood culture and valve material were culture-negative; and (ii) all patients who were rejected as having IE were also negative by PCR, with the exception of 1 patient who had bacteraemia from another source and 5 patients whose blood culture material was believed to contain an environmental contaminant. Direct molecular identification of the aetiological agents responsible for IE from blood culture material may enable specific treatment to commence at an earlier stage of the disease and hence reduce the need for valve replacement. Such a molecular approach may aid in the diagnosis of IE and should therefore be included as an additional major criterion in the Duke's classification scheme.