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The temporal image subtraction technique was applied to bone scintigraphy, using Photoshop (commercially available image processing software) and Morpher (public domain warping software). For the temporal subtraction images, 81 subtraction images (19 cases) were prepared by a method used to subtract the previous images from the current ones. Registration of the current and previous images was performed by manual operation using Photoshop, and warping was done using the warping function of Morpher. In addition, difference images prepared after correcting the distributions of radioactive isotopes of the current and previous images using the count of the pelvic region were also examined. Compared with manual operation, alignment of images by warping improved registration and reduced the generation of pseudo-images of subtraction images. The rate of identification of abnormal accumulation-enhanced regions and subjective evaluation by doctors was improved for warping more than for manual operation. Furthermore, abnormal hot regions, which are difficult to find in film images, could be found in three subtraction images. In addition, it was confirmed that abnormal hot regions become more visible in many cases by preparing subtraction images after correcting the count between images using the count of the pelvic region. Thus, it is suggested that the temporal image subtraction technique in bone scintigraphy enables more accurate observation of enhancement of or changes in abnormal hot regions, which will support diagnostic reading. It is considered that enhancement of or changes in abnormal hot regions will be more accurately understood through further detailed discussion in the future.