Polyomavirus JC (JCV) has been implicated in the etiology of multiple sclerosis (MS), because it causes progressive multifocal leukoencephalopathy (PML), a multifocal demyelinating disease with many microscopical similarities to MS. During childhood, the virus establishes a latent infection in the kidneys, which can be reactivated in immunocompromised patients. During reactivation, the virus is shed in the urine. The kidney is the only known site of latent infection and reactivation. Therefore, excretion of the virus in the urine of MS patients is to be expected, if reactivated JCV is involved in the etiology of MS. We studied urine samples of 53 patients with definitive MS and of 53 controls matched for age and sex. We found no evidence of active JCV infection in MS. The hypothesis of a polyomaviral etiology of MS is not supported by the results of this study.