It has been recently shown that optical imaging (OI) methods can be used to image the in vivo biodistribution of several radiopharmaceuticals labeled with beta or alpha emitters. In this work particular attention has been focused on investigating the weaker optical signal induced by an almost pure gamma emitter like Tc-99m. Visible light emission measurements of a water solution containing Tc-99m were performed using a small animal OI system. A sequence of images was acquired for 24 h in order to study the decay of the luminescence signal. The difference between the luminescence decay half life and well-known Tc-99m half life was equal to 1%. in vivo imaging was performed by injecting one control nude mice with Tc-99m-MDP. Optical images obtained with equipment designed for bioluminescence imaging showed that a visible light emission was distinguishable and correctly localized in the bladder region where a higher concentration of Tc-99m-MDP was expected. The bladder to background ratio was always greater than 1. We conclude that the experimental data presented in this paper show that it is possible to detect in vivo luminescence optical photons induced by Tc-99m. This is important especially considering the large number of Tc-99m–based radiopharmaceutical currently available.