The cause of multiple sclerosis (MS) is currently unknown. The hypothesis of this study is that MS is caused by a chronic bacterial infection of the central nervous system (CNS) and the ensuing immune response. We developed a sensitive two-stage polymerase chain reaction method using nested or semi-nested primers specific for the bacterial 16S ribosomal DNA to test for the presence of bacterial DNA in cerebrospinal fluid. We designed seven sets of primers to amplify DNA from spirochetes , Campylobacter, Mycoplasma, Chlamydia, Bartonella, Mycobacteria and Streptococcus and tested cerebrospinal fluid from patients with relapsing-remitting MS, primary progressive MS, transverse myelitis and controls. We did not detect DNA from any of the groups of bacteria in patients or controls. We conclude that we were unable to find evidence for CNS infection with any of these seven groups of bacteria in MS.