Mycoplasma pneumoniae and Legionella pneumophila are increasingly recognized as important agents of community-acquired lower respiratory tract infections (LRTI). Mycoplasma pneumoniae has been also recognized as a cause of nosocomial infections. The aim of this study was to investigate the role of real time polymerase chain reaction (PCR) for the rapid diagnosis of these infections among hospitalized children with community-acquired LRTI. During 2001, 65 children were prospectively studied. Microbiological investigation consisted of capillary PCR with a LightCycler for M. pneumoniae and L. pneumophila in induced sputum or throat swab specimens, IgM enzyme immunoassay for M. pneumoniae and immunofluorescence for L. pneumophila in paired sera. Serology testing showed acute M. pneumoniae infection in 18 (27.5%) patients and L. pneumophila in 1 (1.5%). M. pneumoniae was also detected in sputum specimen by capillary PCR in 9 (50%) serologically diagnosed cases, including 4 (22%) with non-diagnostic IgM levels in the acute phase. Capillary PCR and IgM enzyme immunoassay diagnosed together 15 (83%) M. pneumoniae cases in the acute phase. It is concluded that M. pneumoniae is an important cause of LRTI necessitating hospitalization among children in Greece. Capillary PCR in sputum may diagnose M. pneumoniae LRTI in the acute setting and direct therapy and isolation of patients.