Invasive pulmonary aspergillosis (IPA) is an important cause of mortality and morbidity in the immunocompromised host. However, the diagnosis of this condition may be difficult, and it is sometimes missed because of the lack of sensitivity of available tests. Therefore, we used polymerase chain reaction (PCR)—based amplification of fragments of genes-encoding alkaline proteases from Aspergillus fumigatus and A. flavus to detect these organisms in bronchoalveolar lavage fluid specimens. The predicted size of the product (747 base pairs) after amplification of A. fumigatus was larger than that for A. flavus (690 base pairs). The reaction was highly sensitive (after amplification of 500 fg of A fumigatus DNA, product could be detected by Southern analysis), and it was specific for A. fumigatus and A. flavus. Bronchoalveolar lavage fluid from four immunosuppressed patients with proved or probable IPA was positive by this assay (sensitivity, 100%); in addition, the sample from one patient with possible IPA was PCR-positive. Only one specimen from 18 immunosuppressed patients with no evidence of IPA was PCR-positive (specificity, 94.4%). Five of 28 bronchoalveolar lavage samples from nonimmunosuppressed patients were PCR-positive, probably representing colonization of the respiratory tract. PCR-based detection may prove useful in the diagnosis of IPA.