Bacteremia and fungemia contribute significantly to the mortality and morbidity of patients. The use of blood cultures is not rapid, sensitive or specific enough to identify or exclude all causes of a febrile episode, resulting in empiric administration of broad-spectrum antibiotics based on clinical grounds. To overcome these limitations, we have made use of the sequence variability of the 16S ribosomal DNA (rDNA) of bacteria and the intergenic transcribed spacer region (ITS2) of fungi for rapid DNA amplification, detection and identification of pathogens from positive blood cultures using an automated fluorescent capillary electrophoresis sequencer. Amplification was tested for cross-reactivity with human DNA, effect of prolonged sample storage and presence of various antibiotics. The SSCP patterns of the most commonly isolated bacteria from blood cultures and the ITS2 length of 47 various fungal species were determined using control strains. The molecular identification of 304 blood culture positive clinical specimens and 101 seeded yeast blood cultures, using the determined SSCP patterns or ITS2 length as reference, was compared to conventional identification in a double-blinded fashion. (Abstract shortened by UMI.)