One of the most effective multiple sclerosis (MS) treatment is natalizumab. Nevertheless, it has been associated with an increased risk of progressive multifocal leukoencephalopathy (PML) caused by the JC virus (JCV). Our main objective was to assess the utility of testing JCV-DNA, apart from anti-JCV antibodies, to determine which natalizumab-treated MS patients has been previously in contact with the virus. For this purpose, 138 MS natalizumab/non-natalizumab treated patients participated in several studies. Cross-sectional study (CS): association of several epidemiological variables with anti-JCV antibodies and JCV-DNA levels in PBMC/serum/urine. First longitudinal study (A): evaluation of JCV-DNA prevalence in urine throughout the treatment. Second longitudinal study (B): simultaneous assessment of antibodies and viral DNA levels in PBMC/serum/urine at two time points. CS: The seropositivity rate for anti-JCV antibodies (62.3 %) and JCV prevalence in urine (59.4 %) were similar; although 26 % of our population was positive only using one of the two techniques. A: The viral prevalence in urine seemed to increase between the baseline visit and the others (Baseline-Visit/V18months, p = 0.006). B: Our rate of positive antibody seroconversion was 36 %. Nearly all patients with detectable JCV-DNA levels in PBMC excreted the virus intermittently in urine; while our PML case, positive in PBMC and serum samples 2 month before the PML, excreted JCV permanently. In conclusion, the determination of JCV DNA levels in urine could be complementary to anti-JCV antibodies for identifying MS patients who has been infected by the JCV. Further research would be necessary to understand the different JCV excretion profiles in urine.