The aim of this study was to evaluate Tc-99m labeled human beta-defensin-1 (HBD-1) for discrimination between bacterial infection and sterile inflammation. For this purpose, HBD-1 was radiolabeled with 99mTc and its in vivo distribution was evaluated in inflamed rats with Staphylococcus aureus (S. aureus) and sterile inflamed rats with turpentine oil. After injection into inflamed and sterile inflamed rats, Tc-99m-HBD-1 was rapidly removed from the circulation via the kidneys. Binding of Tc-99m-HBD-1 to inflamed muscle (T/NT 20 at 120 min) was two times higher than binding to sterile inflamed muscle (T/NT = 10 at 120 min) of rats. It was demonstrated that Tc-99m-HBD-1 can be used to detect S. aureus inflammation in rats. However, the radiolabeled antimicrobial peptide showed only poor uptake in sterile inflammation with turpentine oil in rats. As a result, Tc-99m-HBD-1 can be useful for detection of bacterial inflammation.