We evaluated the value of Aspergillus PCR as a tool for diagnosing invasive aspergillosis from whole-blood samples during antifungal therapy. In a 3-year study, 36 patients receiving antifungal therapy due to chest radiographic findings highly suggestive of fungal pneumonia were evaluated. The PCR results from whole-blood samples were compared to those obtained from bronchoalveolar lavage fluids and/or tissue specimens. A total of 205 whole-blood samples, 15 fine-needle aspirations or tissue biopsy specimens, and 21 bronchoalveolar lavage fluids and tracheal secretions were analyzed using PCR. Of the 36 patients, 15 had proven, 9 had probable, and 12 had possible invasive Aspergillus infection according to European Organization for Research and Treatment of Cancer/Mycosis Study Group definitions. For patients with proven infection the sensitivity values of PCR in lung and blood samples were 100 and 40%, respectively. The negative predictive value of blood monitoring under conditions of antifungal treatment was 44%. Clearance of fungal DNA from blood was associated with resolution of clinical symptoms in six of nine patients with proven infection. Repeated positive PCR results for Aspergillus were associated with fatal outcome, as three of six patients died. For patients with probable infection the sensitivity values of PCR in lung fluid and blood were 66 and 44%, respectively. The benefit of PCR diagnosis using whole-blood samples is limited when sampling takes place after treatment has been started. Performance of Aspergillus PCR using tissue samples is recommended in addition to microscopic examination and culture technique for sensitive detection of fungal infection.