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The purpose of this study was to evaluate the efficacy of digital subtraction radiography using a direct digital imaging system in the follow-up study of endodontically treated teeth. The RVG-S was used as a direct digital imaging system. The intraimage variation of the original RVG-S image caused by dark current and sensitivity variations among pixels was corrected by pixel-to-pixel. The interimage variation was further corrected using a copper step-wedge attached to the sensor. Standardized images were obtained from the same geometrical setup during the follow-up. Pixel values at the regions of interest positioned on the periapical lesion increased after the endodontic treatment, and this change continued during the observation period up to 545 days. The subtraction method with direct digital radiography will be a useful tool to evaluate the healing process in endodontic treatments.