Magnetization transfer imaging (MTI) has been shown to be sensitive to both macroscopic and microscopic disease in multiple sclerosis (MS). In this study three-dimensional MTI was used to estimate the global burden of disease in large volumes of brain tissue. MTI was performed in 15 MS patients and 11 normal controls. In seven MS patients MTI was performed on two different occasions. MTI data were displayed as magnetization transfer ratio (MTR) histograms and analyzed. The peak height of the histograms, presumably reflecting the residual amount of normal brain tissue, was lower in MS patients as compared with normal controls (P < 0.001), and was found to correlate with the duration of disease (P < 0.05). A decrease of the MTR histogram peak height was observed in the course of the disease (P < 0.01). These findings suggest that in MS, volumetric MTI provides quantitative information reflecting the global burden of disease.