BACKGROUND: The role of antimyelin antibodies as biomarker in multiple sclerosis is subject of debate. Here antimyelin antibody reactivity against native myelin is studied in CSF and serum. OBJECTIVE: To compare antimyelin antibody reactivity between patients with multiple sclerosis (MS) and patients with other neurological diseases in CSF and serum. In addition, MRI measures were studied in relation to antimyelin antibody reactivity. METHODS: 77 MS patients (13 primary progressive, 27 secondary progressive and 37 relapsing remitting), 26 patients with other non-inflammatory neurological diseases and 9 patients with inflammatory neurological diseases other than MS were included. A myelin flow cytometry assay was used to detect anti-myelin antibody levels which were expressed as mean fluorescence intensity (myelin-MFI). MRI outcome measures were new or persistent T2 lesions, gadolinium enhancing T1 lesions and brain atrophy which were assessed by normalized brain volumes. RESULTS: There was no significant difference between myelin-MFI values in serum and CSF between MS patients and controls (Mann-Whitney test p=0.19 and p=0.51). Myelin-MFI values in CSF were not correlated with number of T2 lesions (Spearman r=-0.023, p=0.85), number of gadolinium enhancing T1 lesions (Spearman r=-0.066, p=0.588) or normalized brain volume (Spearman r=-0.065, p=0.594). CONCLUSIONS: These results do not confirm an association between anti-myelin antibody reactivity and the presence of MS or MRI measures of disease activity.