The purpose of this in vitro study was to determine the feasibility of using dual wavelength spectrophotometry to identify teeth with pulp chambers that are either empty, filled with fixed pulp tissue, or filled with oxygenated blood. In phase I of the experiment, a human third molar was prepared so that its pulp space could be filled with oxygenated blood and later emptied. In phase II, the lower jaw of a beagle dog was removed and placed in formalin, thereby fixing the pulps of the teeth. The pulp of the right canine was removed via an apical approach, and attachments were placed in a similar position to those on the human tooth, to allow filling and emptying of the pulp space. Cavit was placed over the exposed fixed pulp in the left canine. Ten readings, which were separated by light source and detector removal and replacement, were taken of the right canine pulp space when it was empty or filled with oxygenated blood, or the left canine pulp space when it was filled with fixed tissue. Distinct and reproducible changes were measured for pulp spaces filled with air, tissue, or oxygenated blood. In phase III, simulated pulp testing on a dog tooth model was performed. Blood was introduced into the root canal space, the chamber was rinsed with water and replaced with air, according to a predetermined code. Spectrophotometer readings were recorded. The identification of pulpal contents was correctly determined in all 20 of the predetermined conditions. The findings indicate that continuous wave spectrophotometry may become a useful pulp testing method.