Contamination of hospital water systems with legionellae is a well-known cause of nosocomial legionellosis. We describe a new real-time LightCycler PCR assay for quantitative determination of legionellae in potable water samples. Primers that amplify both a 386-bp fragment of the 16S rRNA gene from Legionella spp. and a specifically cloned fragment of the phage lambda, added to each sample as an internal inhibitor control, were used. The amplified products were detected by use of a dual-color hybridization probe assay design and quantified with external standards composed of Legionella pneumophila genomic DNA. The PCR assay had a sensitivity of 1 fg of Legionella DNA (i.e., less than one Legionella organism) per assay and detected 44 Legionella species and serogroups. Seventy-seven water samples from three hospitals were investigated by PCR and culture. The rates of detection of legionellae were 98.7% (76 of 77) by the PCR assay and 70.1% (54 of 77) by culture; PCR inhibitors were detected in one sample. The amounts of legionellae calculated from the PCR results were associated with the CFU detected by culture (r = 0.57; P < 0.001), but PCR results were mostly higher than the culture results. Since L. pneumophila is the main cause of legionellosis, we further developed a quantitative L. pneumophila-specific PCR assay targeting the macrophage infectivity potentiator (mip) gene, which codes for an immunophilin of the FK506 binding protein family. All but one of the 16S rRNA gene PCR-positive water samples were also positive in the mip gene PCR, and the results of the two PCR assays were correlated. In conclusion, the newly developed Legionella genus-specific and L. pneumophila species-specific PCR assays proved to be valuable tools for investigation of Legionella contamination in potable water systems.