Growing evidence suggests that axonal degeneration rather than demyelination is the pathological substrate underlying chronic, irreversible disability in multiple sclerosis. However, direct evidence linking clinical disability measured in vivo with corresponding post-mortem measures of axonal pathology is lacking. Our objective in this study was to investigate the relationship between motor disability accumulated by patients with multiple sclerosis during life and the degree of axonal loss observed in their descending motor tracts after death. Human spinal cord derived at autopsy from 45 patients with multiple sclerosis was investigated. The medical records of each patient were reviewed by a multiple sclerosis neurologist to determine the degree of motor disability reached before death. Spinal cord sections were stained immunohistochemically. The degree of demyelination and the number of surviving corticospinal tract axons were measured in each patient. Patients who had accumulated higher levels of motor disability prior to death demonstrated fewer surviving corticospinal axons. Motor disability did not correlate with degree of demyelination. This study provides for the first time, direct clinico-pathological evidence that axonal loss is the pathological substrate of established disability in multiple sclerosis.