Although peripheral blood myelin-autoreactive T cells are thought to play a key role in multiple sclerosis, they are generally considered to have qualitative differences rather than quantitative ones when compared to those found in healthy individuals. Here, we revisited the assessment of myelin-autoreactive T cells in a new approach based on their combined ability to acquire membrane proteins from autologous antigen presenting cells, and to respond to whole myelin extract as the stimulating autoantigen. Using this approach, the myelin-autoreactive T cell frequency in patients with multiple sclerosis was found to be unexpectedly high (n = 22, subtracted values median 2.08%, range 0-6%; background median 1%, range 0-4%) and to exceed that of age/gender-matched healthy individuals significantly (n = 18, subtracted values median 0.1%, range 0-5.3%, P < 0.0001; background median 1.45%, range 0.1-4%). Higher anti-myelin autoreactivity was stable in patients with multiple sclerosis after several months. These data correlated with whole myelin-induced gamma interferon-enzyme-linked immunosorbent spot assay performed under the same conditions, although the values obtained with enzyme-linked immunosorbent spot assay under all conditions were 58 times lower than with this new method. The myelin-autoreactive T cells were memory T cells expressing CD40L with a CD62(low) phenotype, suggesting their ability for homing to tissues. Collectively, these new data show a higher frequency of autoreactive T cells during multiple sclerosis than in age/gender-matched healthy individuals, and support an autoimmune aetiology in multiple sclerosis.