The idea that the initiating event in the formation of all new multiple sclerosis lesions is a focal blood-brain barrier (BBB) leakage associated with perivascular inflammation has been challenged recently by the observation of subtle abnormalities in some quantitative magnetic resonance (MR) parameters (including the magnetization transfer ratio) prior to lesion enhancement. MR diffusion imaging can non-invasively quantify the average apparent diffusion coefficient (ADC(av)), a measure of water molecule random motion that is sensitive to pathological change in multiple sclerosis lesions and to abnormalities in the normal-appearing white matter (NAWM). We therefore used MR diffusion imaging to investigate the dynamic evolution of water diffusion measurements in new enhancing multiple sclerosis lesions, in the NAWM from which they arise, and in anatomically matched contralateral NAWM regions from which no visible lesions develop. Gadolinium diethylenetriaminepentaacetic acid (Gd)-enhanced MRI and MR diffusion studies were performed monthly for 1 year in five multiple sclerosis patients with clinically and radiologically active disease. The ADC(av) was calculated at each time point of the study (before, during and after lesion appearance on Gd-enhanced scans) for each new enhancing lesion, and for regions matched for size and position in the contralateral NAWM. A steady and moderate increase in ADC(av) in prelesion NAWM was observed, which was followed by a rapid and marked increase at the time of Gd enhancement and a slower decay after the cessation of enhancement. In matched contralateral NAWM regions there was a significant but milder increase in ADC(av) at the time of the first noted lesion enhancement. These findings indicate that new focal lesions associated with frank BBB leakage are preceded by subtle, progressive alterations in tissue integrity beyond the resolution of conventional MRI. The increases in ADC(av) in anatomically matched contralateral regions after lesions have appeared supports the concept that structural damage in lesions causes damage or dysfunction in connected areas of NAWM.