Two sensitive and specific RT-PCR assays were standardised for testing the presence of human metapneumovirus. A total of 300 nasopharyngeal aspirates collected from infants suffering from bronchiolitis since October 2000 to June 2003 and shown previously as negative to common respiratory viruses were examined. Matrix and polymerase viral genes, which show a low rate of variation, were chosen to design amplification assays to ensure that any genotype of the human metapneumovirus could be detected. A RT-PCR followed by a reverse line blotting hybridisation was developed for viral polymerase gene. For the matrix gene, after the RT-PCR assay, a subsequent nested PCR was carried out. Both assays had similar sensitivity, equivalent to 0.1 TCID50 of human metapneumovirus strain NL/1/99 which was used as the positive control. The human metapneumovirus was present in 16.6% of the specimens studied. The approaches described below are not only a robust method for rapid diagnosis of the human metapneumovirus, but also to establish an etiological surveillance tool for epidemiological studies. Based on the results obtained, human metapneumovirus infections in Madrid followed a seasonal pattern, with most of the infections occurring between February and April.