Serial brain MRI scanning is widely used for assessing multiple sclerosis disease activity in the evaluation of new therapies. Traditionally, the net change in T2-weighted lesion volume between paired scans has been used as a measure of disease progression and as a secondary endpoint in definitive clinical trials. However, as the net change in T2-weighted lesion volume reflects only the difference between new and resolved T2-weighted lesions, this measure significantly under-represents the total T2-weighted lesion activity. Difference images produced by subtracting labelled T2-weighted lesion volumes from serial registered T2-weighted scans allows separate measurements of individual volumes of new and resolving T2-weighted lesions, which may reflect underlying disease activity more sensitively. We generated T2-weighted differences images to define T2-weighted lesion changes over 1 year for 19 patients with relapsing multiple sclerosis. The mean new T2-weighted lesion volume change was three times greater than the mean net T2-weighted lesion volume change over the study period. New T2-weighted lesion volumes were more strongly correlated with T1-weighted gadolinium-enhancing lesion volumes (r = 0.72, P = 0.001) than were the net T2-weighted lesion volume changes (r = 0.45, P = 0.01). Baseline T2-weighted lesion volume was more highly correlated with new T2-weighted lesion volumes (r = 0.89, P < 0.0001) than with net T2-weighted lesion change (r = 0.47, P < 0.001). There was a trend for patients who showed sustained clinical progression over the year to have a greater new T2-weighted lesion volume than those who did not. This difference was not seen with net T2-weighted lesion volume change. T2-weighted lesion difference images should provide an additional and sensitive tool for monitoring disease activity in multiple sclerosis. Independent definition of new and resolving T2-weighted lesion volumes also offers the potential for discrimination of the relative effects of experimental therapies on new inflammatory activity from the effects on oedema resolution and lesion repair.