The Optic Neuritis Treatment Trial (ONTT) is a prospective study of corticosteroid treatment of acute optic neuritis (ON), with subsequent longitudinal follow-up to determine development of clinically definite multiple sclerosis (CDMS). We analyzed the CSF of 83 patients with clinically isolated ON who underwent lumbar puncture within 24 hours of enrollment into the ONTT to determine the value of CSF changes in ON, especially regarding diagnostic utility, immunologic changes, MRI correlations, and progression to CDMS. All patients had baseline MRI scans graded for changes typical of MS. CSF measurements included immunoglobulin G (IgG) synthesis, IgG ratio, myelin basic protein, IgG kappa light chains, and oligoclonal banding. No patients had their diagnosis or management altered as a result of CSF findings. Except for oligoclonal bands, few patients showed any abnormalities on CSF tests, and no tests correlated with the 2-year development of CDMS. Oligoclonal banding, present at baseline in 11 of 13 patients who developed CDMS, did predict progression to CDMS, but this was not independent of MRI abnormalities. Two patients with oligoclonal bands and a normal MRI did progress to CDMS. We conclude that CSF analysis may not be necessary in the routine evaluation of patients presenting with a typical clinical profile of acute ON, and that most CSF tests add little additional information to MRI results for predicting the 2-year development of CDMS. However, the precise role of oligoclonal banding in the analysis of such patients awaits longer follow-up of this cohort.