A previous study of the sensitivity and accuracy of a standardized radiographic technique for the disclosure of root cavities in a cadaver material indicated that it was not possible to perceive small artificial "resorption" cavities. Using the same material, the aim of the present study was to evaluate whether the use of subtraction radiography would improve the detection of root cavities. In an autopsy material of five mandibular blocks each containing two premolars, small, medium, and large cavities were drilled in the cervical, middle, and apical thirds of the proximal and oral root surfaces. Each jaw block was radiographed before (B) and after (A) cavity preparation with three different exposure times resulting in light, medium, and dark film densities. The radiographs were digitized. Digital subtraction was performed between the A- and B images of varying densities by a computer program developed for dental subtraction radiography. Conventional and reverse contrast modes of the subtraction image were assessed independently by four observers. The results showed small inter- and intraobserver differences in diagnostic accuracy for assessment of total number of root cavities. Original film density did not influence the diagnostic performance in the resulting subtraction image. Contrast mode in the subtraction image influenced diagnostic performance as the majority of observers did best with the reverse contrast mode (p < 0.05). Some of the small root cavities were disclosed by the subtraction technique, but overall accuracy was not increased compared to conventional radiography.