During October 1999 to November 2000, 98 blood culture specimens from the same number of febrile episodes originating from 49 patients with haematological malignancies were examined for the presence of eubacteria and fungi based on 16S rRNA gene and the 5.8, 18 and 28S rRNA combined with in vitro PCR amplification and sequencing, in addition to conventional blood culture laboratory techniques. Nineteen of the samples were associated with positive blood cultures. Eubacterial (16S rRNA) PCR detected bacterial DNA in 26 febrile episodes, i.e. in an additional seven febrile episodes than blood-culture alone. The species identified by partial 16S Rrna gene sequencing were as follows Staphylococcus spp (n=6), Staphylococcus epidermidis (n=5), Acinetobacter spp (n=5), Escherichia coli (n=2), Enterobacter agglomerans (n=2), Campylobacter spp (n=1), Citrobacter spp (n=1), Corynebacterium spp (n=1), Enterobacter faecium (n=1), Ralstonia spp (n=1), Acidovorax spp. (n=1) and Stenotrophomonas maltophilia (n=1). Gram-positive bacteria were found in 12/27 (44.6%) and Gram-negative bacteria were found in 15/27 (55.6%). After optimization of a PCR-based fungal detection method, none of the febrile episodes were shown to be attributable to fungi. The results of this study suggest that fungi are not common causal agents of febrile episodes in patients with a haematological malignancy at this centre and that molecular techniques can augment cultural methods in the diagnosis of causal agents of bacteraemia in patients, so that appropriate antibiotic regimens may be commenced in patients with culture-negative episodes of infection.