Purpose : To develop a new subtraction program for registering digital periapical images based on the correspondence of anatomic structures. Materials and Methods: The digital periapical images were obtained by Digora system with Rinn XCP equipment after translation of 1-16 mm, and rotation of 2-20° at the premolar and molar areas of the human dried mandible. The new subtraction program, NIH Image program and Emago/Advanced program were compared by the peak-signal-to noise ratio (PSNR). Results : The new subtraction program was superior to NIH Images program and Emagol Advanced program up to 16 mm translation and horizontal angulation up to 4°. Conclusion: The new subtraction program can be used for subtracting digital periapical images.