A cohort of 50 consecutive patients with acute monosymptomatic optic neuritis (ON) from a defined catchment area joined a prospective study. The aim of this study was to compare the sensitivity of magnetic resonance imaging (MRI), electrophysiological methods (VEP and SEP) and biothesiometry to detect abnormalities in other parts of the CNS than the optic nerves during the acute phase of ON. For each method, a scoring system is proposed. This investigation also hoped to achieve a better understanding of the natural history of ON. MRI proved to be the most sensitive tool (63% abnormal) in confirming a second site of involvement, followed by VEP in the clinically unaffected fellow eye (42%), biothesiometry (32%) and SEP (17%). The combination of all these methods, except for MRI (and VEP in eyes with acute ON), revealed abnormalities in 63% of the patients. When the neurophysiological methods were combined with MRI, 79% of the patients had abnormal findings suggesting additional lesions in the CNS. Hence, MRI and neurophysiological examinations supplement each other and together provide evidence that monosymptomatic ON is usually a first manifestation of MS. The development of definite MS at 1-20 months of follow up in 7 patients (all with abnormal MRI initially) supports this view.