Purpose: To compare the standard deviation of gray levels on digital subtracted images obtained by different dental subtraction programs. Materials and Methods: Paired periapical films were taken at the lower premolar and molar areas of the phantoms involving human mandible. The bite registration group used Rinn XCP equipment and bite registration material, based on polyvinyl siloxane, for standardization. The no bite registration group used only Rinn XCP equipment. The periapical film images were digitized at 1200 dpi resolution and 256 gray levels by a flat bed scanner with transparency unit. Dental digital subtraction programs used for this study were Subtractor (Biomedisys Co., Korea) and Emago (Oral Diagnostic Systems, The Netherlands). To measure the similarities between the subtracted images, the standard deviations of the gray levels were obtained using a histogram of subtracted images, which were then analyzed statistically. Results: Subtracted images obtained by using the Emago program without manual selection of corresponding points showed the lowest standard deviation of gray levels (p<0.01). And the standard deviation of gray levels was lower in subtracted images in the group of a bite registration than in the group of no use of bite registration (p < 0.01). Conclusion: Digital radiographic subtraction without manual selection of reference points was found to be a convenient and superior method.