Proton magnetic resonance spectroscopy has shown elevated signals in the spectral region of lipids in acute multiple sclerosis lesions. The metabolite-nulling technique allows the separation of macromolecules from other metabolites, such as lactate, N-acetyl-aspartate, creatine, choline and myo-inositol. Using this technique in studies on multiple sclerosis patients, we were able to differentiate macromolecules biochemically in acute and chronic multiple sclerosis lesions. Ten patients with acute, contrast-enhancing multiple sclerosis lesions, 10 patients with chronic lesions and 10 healthy control subjects were investigated with a 1.5 T whole body system, using a stimulated echo acquisition mode (STEAM) sequence with metabolite-nulling and outer volume saturation. Metabolites and macromolecules were quantitated absolutely. The 0.9 and 1.3 parts per million (p.p.m.) resonances of the macromolecules were significantly elevated in acute lesions compared with chronic lesions and healthy controls (P < 0.001 for 0.9 p.p.m., P < 0.05 for 1.3 p.p.m.). The macromolecular resonances at 2.1 and 3.0 p.p.m. in acute and chronic lesions were normal. N-acetyl-aspartate was significantly reduced in acute and chronic lesions compared with controls (P < 0.05 and P < 0.01, respectively). Choline was significantly elevated in acute lesions compared with controls (P < 0.05). Up to now, elevated resonances at 0.9 and 1.3 p.p.m. in acute lesions have been interpreted as lipids. In metabolite-nulled spectra, the macromolecular resonances did not fit those of lipids and might have been due to proteins or polypeptides containing the amino acids alanine, threonine, valine, leucine and isoleucine. These account for approximately 40% of the amino acids of myelin proteolipid protein and for approximately 20% of myelin basic protein. The increased macromolecular resonances at 0.9 and 1.3 p.p.m. may be interpreted as biochemical markers of myelin fragments and may be used as reliable markers of acute multiple sclerosis lesions as they provide clear discrimination among acute and chronic lesions and controls.