In vivo optical imaging is widely used in preclinical studies. Recently, the application of optical imaging systems for preclinical visualization of gamma-emitting isotopes has become of interest since the evaluation of various organs relies on Tc99m-based radiopharmaceuticals (RPs). In vitro radioluminescence of Tc99m-based RPs, including pertechnetate, albumin macroaggregates, dimercaptosuccinic acid, phytate colloid, and ethylenediamine tetramethylene phosphonic acid, was studied with IVIS Spectrum CT™ optical imaging system. The distribution of phytate colloid was also studied in vivo with and without scintillating materials and the results were compared with those obtained with a conventional scintigraphy. The visible light emission appeared to be due to the radioluminescence of water and luminophores contained in RPs rather than from Cherenkov radiation. Weak air luminescence affected the background. The radioluminescence of fluids induced by Tc99m-based tracers could be detected using charge-coupled device optical imaging systems. The radioluminescence intensity and its spectral distribution depend on the surrounding fluid and known luminophores present. Thus, in some cases the in vivo optical imaging is possible but the use of scintillator, e.g., borosilicate glass or bismuth germanate, is preferred.