To compare noise in subtraction images produced by two subtraction programs. Twenty-five pairs of bitewing radiographs exposed under clinical conditions were subtracted using two digital subtraction programs: X-PoseIt (XP) software and EMAGO/Advanced 3.43 (EM) software. Four reference points were located between the mesial surface of the upper first premolar and the distal surface of the upper first molar (the region of interest) on the same anatomical structures using XP and EM. The standard deviation (SD) of the histogram defining the distribution of the shades of grey in the region of interest was used as the statistical parameter for comparing noise in the subtraction images created by the two subtraction programs. The mean and median grey shade value was lower with EM software (126.9, 126) than with XP software (128.3, 128) (P < 0.05). For the SD, the mean value was significantly lower with XP (6.2) than with EM (7.8) (P < 0.05). The range of the SD values was larger for EM (3.6-12.8) than XP (3.3-9.9). The standard deviation of the grey shade histogram showed that subtraction images produced with X-PoseIt software were statistically less noisy than images produced with EMAGO/Advanced 3.43 software.34357-61