Invasive aspergillosis is associated with high morbidity and mortality rates; nevertheless, blood cultures almost invariably yield a negative result. The recovery and detection time of Aspergillus fumigatus, Aspergillus flavus and Aspergillus terreus were studied in BACTEC Plus Aerobic/F, Mycosis-IC/F and Myco/F Lytic vials, incubated in the BACTEC 9240 and 9000 MB automated systems. Two different approaches were used for subculture in solid medium: (i) the routine method, using a sterile airway needle/subculture unit, and (ii) a novel procedure, using instead a tuberculin disposable syringe and collecting a larger aliquot (100 µl), following vigorous agitation of the vials. A. fumigatus was detected at inoculum concentrations of >3 conidia per 10 ml after 21-40 h, in both BACTEC Plus Aerobic/F and BACTEC Mycosis-IC/F vials. A few more hours were needed to detect A. flavus and A. terreus. The novel subculture procedure of BACTEC culture vials on solid medium resulted in several positive results that were not detected by the routine sampling procedure. BACTEC Plus Aerobic/F vials show an advantage particularly in patients under antifungal treatment. In cases of polymicrobial bloodstream infections (concurrent bacterial growth), the inoculation of blood samples into a BACTEC Mycosis-IC/F vial achieved the best results. Further multicentre studies are needed to validate this improved automated detection of Aspergillus spp. from blood cultures in clinical laboratories, as this diagnostic procedure allows antifungal susceptibility testing of moulds.