Natalizumab-associated progressive multifocal leukoencephalopathy in multiple sclerosis (MS) occurred in two individuals also treated with interferon β1a, raising concerns about the interaction of these disease-modifying agents and leading to the recommendation to avoid their concomitant administration. However, type I interferons are antiviral. Using a real-time quantitative polymerase chain reaction for the detection and quantification of the John Cunningham virus (JCV), DNA in peripheral blood mononuclear cells (PBMCs), and urine in MS patients, we tested the hypothesis that MS disease-modifying drugs (DMD) qualitatively and quantitatively alter JCV prevalence and viral copy numbers. Two hundred thirty-nine patients were enrolled in a cross-sectional study in which blood and urine specimens were collected at a single time and 37 newly diagnosed, treatment-naïve MS patients were enrolled in a longitudinal study in which specimens were obtained at diagnosis and 6 months after treatment initiation. JCV DNA was detected in PBMCs of only two patients (0.07 %), but was commonly detected in the urine (46.8 %) in this population. There was no effect of DMDs on blood or urinary JCV prevalence or viral copy numbers with either glatiramer acetate (Copaxone®) or interferon-β therapy (Avonex®, Betaseron®, or Rebif®). The small number of patients on other therapies precluded meaningful comment about their effects. No obvious effect of the platform DMDs on JCV prevalence was observed even for the interferon-βs.