Background: Natalizumab-related progressive multifocal leukoencephalopathy (PML) is associated with the presence of anti-John Cunningham virus (JCV) antibodies. The aim of this investigation was to evaluate the long-term stability of anti-JCV antibody serum levels and their relation to various demographic, clinical and radiological characteristics in patients suffering from multiple sclerosis (MS). Methods: Seventy-eight relapsing-remitting MS patients treated with natalizumab and evaluated for the presence of serum anti-JCV antibodies over a time period of 1-6 years (3-11 samples) were included in the study. Anti-JCV antibody levels and their changes were correlated with various demographic, clinical and radiological findings. Results: Median follow-up time was 43.5 months, with a median of 5.3 samples available per patient. At baseline, 46 (59%) of the patients were seropositive. During follow-up, anti-JCV antibody status changed from negative to positive or vice versa in 23% of patients. Baseline anti-JCV antibody index correlated positively with age (p = 0.03). Patients: with stable positive anti-JCV antibody index had more T2 hyperintensities (20.2 vs. 13.1; p < 0.007) on cerebral magnetic resonance imaging (MRI) and were also older than the stable negative anti-JCV antibody index group of patients (45.2.vs. 40.3 years; p < 0.01). No significant increase in T2 hyperintensities after seroconversion was revealed. Average duration from beginning of natalizumab therapy to seroconversion fit = 13) was 33 months. Annual seroconversion rate of anti-JCV antibody status was 6.5%. A baseline anti-JCV antibody index of > 0.90 (tt = 33) predicted stable seropositivity (100%), while baseline anti-JCV antibody index <0.20 did not predicted stable seronegativity (59%). PML was not diagnosed in any of the patients studied during the follow-up. Conclusions: A positive baseline anti-JCV antibody index of > 0.90 predicts stable positive JCV serostatus, in contrast with a baseline anti-JCV antibody index of <0.20, which remained negative in 59% of cases. Stable positive anti-JCV index patients have more MRI T2 hyperintensities and are older compared with stable negative anti-JCV index patients.