Radiolabeled antibiotics are promising radiopharmaceuticals for the precise diagnosis and detection of infectious lesions. Doxycycline Hyclate (DOX) was chosen to investigate new Tc-99m-labeled antibacterial agent. Ready to use freeze dry kits were formulated with optimum labeling conditions. Human serum stability, sterility, and pyrogenicity of kits were estimated, and gamma scintigraphy, in vivo biodistribution, and histopathological studies with bacterial infected rats were performed. DOX were successfully labeled by Tc-99m with high radiochemical purity, and the labeled compound was stable in human serum. Kits were sterile, pyrogen-free, and stable up to 6months. Static images depicted rapid distribution throughout the body and high uptake in bacterial infected thigh muscle. The uptake ratios of radiopharmaceuticals in infected thigh muscle were found above 2 up to 5h. Five hours after injection, the rats were sacrificed, and biodistribution was determined. Samples of bacterial infected muscle, healthy muscle, blood, liver, spleen, lung, kidney, stomach, intestine, urine and heart were weighed, and the radioactivity was measured by using a gamma counter. The %ID/g of Tc-99m-DOX was found 0.230.06 for infected thigh muscle. According to the imaging, biodistribution, and histopathological studies, the promising characteristics of Tc-99m-DOX make the new radiopharmaceutical valuable to examine for future studies. Copyright (c) 2013 John Wiley & Sons, Ltd.