We present a method for brain single photon emission computed tomography (SPECT) analysis based on individual registration of anatomical (CT) and functional (133Xe regional cerebral blood flow) images and on the definition of three-dimensional functional regions of interest. Registration of CT and SPECT is performed through adjustment of CT-defined cortex limits to the SPECT image. Regions are defined by sectioning a cortical ribbon on the CT images, copied over the SPECT images and pooled through slices to give 3D cortical regions of interest. The proposed method shows good intra- and interobserver reproducibility (regional intraclass correlation coefficient approximately 0.98), and good accuracy in terms of repositioning (approximately 3.5 mm) as compared to the SPECT image resolution (14 mm). The method should be particularly useful for analysing SPECT studies when variations in brain anatomy (normal or abnormal) must be accounted for.