The aim of this study is to radiolabel ganciclovir, known as having selective antiviral properties against thymidine kinase, with technetium tricarbonylcore (Tc-99m(CO)(3)(+)) and to investigate the biological behavior of this complex in vitro and in vivo. Commercially provided Ganciclovir (GCV) was radiolabeled with Tc-99m(CO)(3)(+). Initially, optimum radiolabeling conditions were determined by analyzing factors such as temperature, pH and time. Quality control of the radiolabeled compound was performed. The radiolabeling yield was found to be 97%. The Tc-99m(CO)(3)-GCV complex also displayed good in vitro stability during the 24 h period. In vitro cell uptake studies showed that the Tc-99m(CO)(3)-GCV complex is highly uptaken in A-549, PC-3, HeLa cell lines according to the control group Tc-99m(I)-tricarbonyl core. The knowledge gained from in vivo and in vitro studies of Tc-99m(CO)(3)-GCV could contribute to the development of a new HSV1-tk gene imaging agent.