OBJECTIVE. Image registration and subtraction to detect the change of disease burden in multiple sclerosis on serial MR images should benefit from the use of high-resolution isotropic voxels. We compared 1.2-mm isotropic three-dimensional (3D) fast fluid-attenuated inversion recovery (FLAIR) images with standard 3-mm two-dimensional spin-echo images for the detection of new or enlarging lesions in longitudinal studies. SUBJECTS AND METHODS. Serial MR images were obtained at baseline, month 6 (n = 20), and month 7 (n = 16). For the half-yearly intervals, subtracted 3D FLAIR images and T2-weighted spin-echo images were compared. For the monthly intervals, subtracted 3D FLAIR images were compared with triple-dose contrast-enhanced T1-weighted spin-echo images. New, enlarging, and enhancing lesions were marked in consensus by two radiologists. RESULTS. At the half-yearly intervals, 3D FLAIR imaging detected more new or enlarging lesions than T2-weighted spin-echo imaging, both at the initial interpretation (80 vs 52; p < 0.001) and after a side-by-side comparison of the lesions (88 vs 65; p < 0.001). Post hoc analyses showed the largest benefit for new (rather than enlarging), for small, and for temporal lesions. At the monthly intervals, 32 enhancing lesions were detected on contrast-enhanced T1-weighted spin-echo images versus 20 new or enlarging lesions detected on 3D FLAIR images (p < 0.05). After a side-by-side comparison of the lesions, seven additional lesions were identified on 3D FLAIR images, making the difference with contrast-enhanced T1-weighted spin-echo images insignificant (27 vs 32; p > 0.05). CONCLUSION. Isotropic 3D FLAIR imaging holds great promise for the detection of new or enlarging lesions in multiple sclerosis using registration and subtraction techniques certainly at longer intervals.