A PCR assay was developed for the detection and identification of Candida and Aspergillus species. The design of the oligonucleotide primer pair as well as the species-specific probes used for species identification was derived from a comparison of the sequences of the 18S rRNA genes of various fungal pathogens. The primers targeted a consensus sequence for a variety of fungal pathogens. The assay was tested for sensitivity and specificity with 134 fungal and 85 nonfungal isolates. To assess clinical applicability, 601 blood samples from four defined groups were tested: group A (n = 35), controls; groups B to D (n = 86), patients with febrile neutropenia, without fungal colonization (group B; n = 29) and with fungal colonization (group C; n = 36); and patients with documented invasive fungal infection (IFI) (group D; n = 21). The assay detected and, by species-specific hybridization, identified most of the clinically relevant Candida and Aspergillus species at 1 CFU/ml of blood. Amplification was 100% sensitive for all molds and yeasts tested, with Histoplasma capsulatum being the only non-Aspergillus species hybridizing with the Aspergillus spp. probe. None of 35 group A patients and only 3 of 65 group B and C patients were PCR positive. The sensitivity of the assay for specimens from patients with IFI (21 patients in group D) was 100% if two specimens were tested. For specificity, 3 of 189 specimens from patients at risk but with negative cultures were positive by the assay, for a specificity of 98%. PCR preceded radiological signs by a median of 4 days (range, 4 to 7 days) for 12 of 17 patients with hepatosplenic candidiasis or pulmonary aspergillosis. For the 10 patients with IFI responding to antifungal therapy, PCR assays became persistently negative after 14 days of treatment, in contrast to the case for 11 patients, who remained PCR positive while not responding to antifungal therapy. Thus, the described PCR assay allows for the highly sensitive and specific detection and identification of fungal pathogens in vitro and in vivo. Preliminary data from the screening of a selected group of patients revealed some value in the early diagnosis and monitoring of antifungal therapy.