We have estimated the accuracy of volume measurements of multiple sclerosis (MS) lesions made using magnetic resonance imaging (MRI) for lesions of comparable diameter to the image slice thickness. We used a phantom containing objects of known volume and obtained images using a range of slice thicknesses. Measurements on the phantom were used to assess a theoretical model, which was then employed to investigate the effects of image dimensions and geometry upon volume measurement accuracy. We observed measured volume to be dependent upon slice thickness. Thin slices gave the most accurate estimate of volume. As slice thickness increased relative to object diameter, the error in the volume measurement increased (to as much as 100%), the volume measured being dependent on the position of the object relative to the slice center. Using a signal intensity threshold value of 50% to outline objects gave results closest to the actual volume. As expected, a lower threshold value tended to give higher volume estimates (up to 100% larger), as did a semi-automated local edge detection technique. For accurate volume measurement, the slice thickness should be no more than a fifth of anticipated object diameter. For typical MS lesions (7 mm in diameter), this implies using a 1.5-mm slice thickness. For serial studies, a repositioning error of 1 mm could lead to differences in the volume measurement of individual lesions of up to 12% between studies for lesions of typical MS size and 5-mm slice thickness. These results emphasize the need for accurate patient repositioning, relatively thin slices, for regular quality assurance checks to ensure that pixel size and slice position are correct and stable over time, and that lesion outlining is performed in a consistent fashion. We would recommend the use of a 3D sequence with 1 mm cubic voxels for accurate measurements of MS lesions.