Twenty-seven patients with acute monosymptomatic optic neuritis were randomly selected from a population-based cohort of patients extensively screened for known etiologies of ON. Paired serum and CSF obtained median 20 days from onset were examined for oligoclonal IgG, free kappa and free lambda chains, and virus-specific oligoclonal IgG antibodies by an affinity-mediated capillary blot technique. CSF-restricted oligoclonal IgG bands, free kappa and free lambda chain bands were observed in 17, 15 and nine patients, respectively. In addition, 16 patients showed a polyspecific intrathecal synthesis of oligoclonal IgG antibodies against one or more viruses (12 measles, nine varicella zoster, six rubella, six mumps) compared to all the 18 examined patients with definite multiple sclerosis (P = 0.0014). The presence of virus-specific oligoclonal IgG was significantly related to the results of oligoclonal IgG (P = 0.0034), free kappa chain bands (P = 0.0020), and brain MRI abnormalities (P = 0.0402). At 1 year follow-up five patients had developed clinically definite multiple sclerosis; all had virus-specific oligoclonal IgG antibodies, oligoclonal IgG and abnormal MRI at onset.