We developed a rapid thermocycling, real-time detection (also known as real-time PCR) method for the detection of Legionella species directly from clinical specimens. This method uses the LightCycler (Roche Molecular Biochemicals, Indianapolis, Ind.) and requires approximately 1 to 2 h to perform. Both a Legionella genus PCR assay and Legionella pneumophila species-specific PCR assay were designed. A total of 43 archived specimens from 35 patients were evaluated, including 19 bronchoalveolar lavage (BAL) specimens and 24 formalin-fixed, paraffin-embedded open lung biopsy specimens. Twenty-five of the specimens were culture-positive for Legionella (9 BAL specimens and 16 tissue specimens). BAL specimens were tested by LightCycler PCR (LC-PCR) methods and by a direct fluorescent antibody (DFA) assay, which detects L. pneumophila serogroups 1 to 6 and several other Legionella species. Tissue sections were tested by the two LC-PCR methods, by DFA, by an in situ hybridization (ISH) assay, specifically designed to detect L. pneumophila, and by Warthin-Starry (WS) staining. The results were compared to the "gold standard" method of bacterial culture. With BAL specimens the following assays yielded the indicated sensitivities and specificities, respectively: Legionella genus detection by Legionella genus LC-PCR, 100 and 100%; Legionella genus detection by DFA assay, 33 and 100%; and L. pneumophila detection by L. pneumophila species-specific LC-PCR, 100 and 100%. With open lung biopsy specimens the following assays yielded the indicated sensitivities and specificities, respectively: Legionella genus detection by LC-PCR 68.8 and 100%; Legionella genus detection by DFA assay, 44 and 100%; Legionella genus detection by WS staining, 63 and 100%; L. pneumophila species-specific detection by LC-PCR, 17 and 100%; and L. pneumophila species-specific detection by ISH, 100 and 100%. The analytical sensitivity of both LC-PCR assays was <10 CFU/reaction. LC-PCR is a reliable method for the direct detection of Legionella species from BAL specimens. The Legionella genus LC-PCR assay could be performed initially; if positive, L. pneumophila species-specific LC-PCR could then be performed (if species differentiation is desired). The speed with which the LC-PCR procedure can be performed offers significant advantages over both culture-based methods and conventional PCR techniques. In contrast, for the methods evaluated, culture was the best for detecting multiple Legionella species in lung tissue. WS staining, Legionella genus LC-PCR, and L. pneumophila species-specific ISH were useful as rapid tests with lung tissue.