Guidelines for the use of polymerase chain reaction (PCR)-based assays to aid the diagnosis of invasive aspergillosis (IA) in high-risk haematology patients have not been formulated. We prospectively evaluated a nested PCR assay to detect Aspergillus in blood during 95 febrile neutropenic episodes, in patients with haematological malignancy and haematopoietic stem cell transplant (HSCT) recipients. PCR results were correlated with the diagnostic classification of the 2002 European Organisation for Research and Treatment of Cancer/Mycosis Study Group. When two-positive results were used to define an episode as 'PCR positive', the sensitivity, specificity, positive-predictive value and negative predictive value for 'proven'/'probable' IA (n = 13) were 100%, 75.4%, 46.4% and 100%, respectively. Consecutive positive results occurred in 61.5% of these 13 episodes. Overall, PCR positivity preceded standard diagnosis by a mean of 14 d and the median time between positive results was shorter than that in other categories of IA. All 13 episodes occurred in the setting of allogeneic HSCT recipients and acute leukaemia. If 'eligibility' for antifungal therapy were based on two-positive-PCR tests, use of empiric treatment could have been reduced by up to 37%. The nested PCR assay is a practical screening test for excluding IA. Patients with consecutive positive results or intermittent-positive results (within 14 d) warrant immediate investigations for IA and the initiation of antifungal therapy.