To investigate the possible association of enterovirus (EV) infection with the development of multiple sclerosis (MS). The role of various genetic and environmental factors has been intensively studied in the etiopathogenesis of MS but the cause of the disease has remained unknown. Enteroviruses are possible candidates because they are neurotropic and able to cause chronic infections. Serum and cerebrospinal fluid (CSF) were tested with reverse transcription-polymerase chain reaction specific for enteroviruses in 17 MS twin pairs. No enteroviral RNA was found in any serum (n = 34) or CSF (n = 12) sample. We found no evidence of enterovirus infection in twins with MS or their healthy siblings. To our knowledge this is a first study to assess the role of enterovirus infections in the risk of developing MS in twins.