Objective The aim of this study was to examine the potential of accurate image registration for detecting subtle changes in the brain. Materials and Methods Isotropic T1-weighted volume images were obtained in 10 normal subjects and five patients on two or more occasions (including pre- and postcontrast studies). The images were segmented and a 3D rigid body translation and rotation technique was used with sine interpolation to precisely match the images using a x2-test. The registered images and the subtraction images produced from them were used to detect changes in signal intensity, site, shape, and size of the brain. Results Small changes due to differences in orientation of the head, growth, and development as well as inhalation of oxygen and carbogen (95% O2/5% CO2) were observed in normal subjects. Changes were also observed in patients with minor head trauma, a meningioma, an astrocytoma, and multiple sclerosis. Differences due to contrast enhancement and surgery and/or anesthesia were also seen. Conclusion With use of subvoxel registration, subtle changes in the brain were detected in a variety of physiological and clinical situations where differences have hitherto been difficult or impossible to detect.