A retrospective study investigating all the infectious encephalitis cases hospitalized at the pediatric intensive care unit of Edouard Herriot University Hospital in Lyon, France, was carried out in order to estimate the prevalence of Mycoplasma pneumoniae in acute childhood encephalitis. From January 2001 to December 2005, the cases of 29 children were selected and reviewed. M. pneumoniae related encephalitis was considered as probable in five cases (17%) on the basis of positive serological tests or positive polymerase chain reaction (PCR) tests in throat or nasopharyngeal swab while the PCR tests performed from the cerebrospinal fluid were negative. This study suggests that M. pneumoniae may be a major cause of infectious encephalitis in children as well as enteroviruses or Epstein-Barr virus detected in five and three cases, respectively.