Hypointense T1 lesions in multiple sclerosis patients correlate with axonal loss at autopsy and biopsy. We evaluated the chemical substrate of hypointense T1 lesions by using in vivo proton magnetic resonance spectroscopy, and analyzed the spectroscopic correlate of increased T1-relaxation time measurements. Localized proton magnetic resonance spectroscopy and T1-relaxation time measurements were performed in lesions, selected on T1-weighted spin-echo magnetic resonance images according to degree of hypointensity, in normal appearing white matter (NAWM) and in normal white matter of controls. In NAWM, prolongation of T1-relaxation time and a decrease in N-acetylaspartate (NAA) were present, compared with normal white matter. Severely hypointense lesions showed a lower concentration of NAA and creatine compared with NAWM and a lower concentration of NAA compared with isointense to mildly hypointense lesions. NAA concentration correlated with degree of hypointensity of lesions and with T1-relaxation time within the spectroscopic voxel. Our results provide the first in vivo evidence of axonal damage in severely hypointense T1 lesions in multiple sclerosis patients. T1-relaxation time correlates with the concentration of NAA in both multiple sclerosis lesions and NAWM, indicating that this parameter deserves further evaluation to monitor disease progression.