Mycoplasma pneumoniae and viruses in acute respiratory tract infections in children were studied during the winter of 1992-1993 in Antwerp, Belgium. M. pneumoniae was diagnosed in nasopharyngeal aspirates by culture and polymerase chain reaction (PCR). For this, amplification of a fragment of the PI adhesin gene in samples prepared by two methods was compared in two laboratories, and in one laboratory, a fragment of the 16S rRNA gene was amplified. The sensitivity of culture versus PCR was 61.5%. Provided a specific internal control is used, sample preparation by freeze-boiling combined with PCR for the PI gene and amplicon detection by visual inspection of the electrophoresis gel can be recommended, although maximal results are obtained after hybridization. M. pneumoniae was present in 0.5% of patients <2 years old and 6.9% of patients >2. M. pneumoniae was second to respiratory syncytial virus or detected equally in lower respiratory infections.