We compared a real time-nucleic acid sequence-based amplification (RTi-NASBA) with conventional NASBA, galactomannan enzyme immunosorbent assay (GMEIA), and Mycology Study Group of the European Organization for Research and Treatment of Cancer (EORTC/MSG) criteria for the diagnosis of invasive aspergillosis (IA). From May 2004 to May 2005, blood samples (314 in total) were collected twice a week from 78 patients with hematologic diseases during neutropenic fever after chemotherapy or hematopoietic stem cell transplantation. Results were compared with each other on the basis of EORTC/ MSG criteria. The cutoff of conventional NASBA was set to be 3.5; GM 0.5; RTi-NASBA, 20% above the negative control. There were 22 patients with IA (7 probables and 15 possibles) and 56 patients with nonfungal infection. The Kappa statistic for RTi-NASBA versus conventional NASBA was 0.80 (0.66-0.82; p<0.001) indicating that there was fairly good accordance between two tests. RTi-NASBA showed sensitivity 0.96, specificity 0.43, positive- and negative-predictive value 0.40 and 0.96, respectively. GM showed good specificity (0.98), while the sensitivity (0.45) was poor. When we use the combination of GM with either of two NASBAs, the sensitivity was improved up to 100%. In conclusion, RTi-NASBA could be a good alternative to the conventional one for the screening of IA.