In the present article, we focused on the radiolabeling and evaluation of Tc-99m-TMH complex as a potential candidate for infection imaging in vivo. For this; Trimethoprim (TMH) used to treat bacterial infections was investigated to label with Tc-99m. Labeling was performed using thin (II) chloride as a reducing agent at room temperature for 1 h and radiochemical analysis involved thin layer radiochromatography (TLRC) and high pressure liquid radiochromatograpy (HPLRC) methods. The stability of labeled antibiotic was checked in the presence of rat blood serum at 37 degrees C up to 180 min. The maximum radiolabeling yield was found to be 96 +/- 2% and remained constant at more than 85 +/- 1% even in rat serum for 180 min after radiolabeling. Static image of Tc-99m-TMH in male rats demonstrated that important radiation signals are present in the infected site at first glance in 30 min. After 30 min the uptake of the Tc-99m-TMH as ID/g% in the infected muscle (INM) and normal muscle (NM) of the rats were 7.5 +/- 1.5% and 5.00 +/- 1.2%, respectively. In the INM/NM ratio a desirable behavior was observed as the values for the INM/NM increased up to 10.6. Tc-99m-TMH prepared with high yield is able to localize well in the bacterially infected muscle of the rats. As a result, Tc-99m-TMH may be developed as a radiopharmaceutical agent to distinguish infection from inflammation by nuclear imaging.