The radiolabeled antibiotic peptide (99m)Tc-alafosfalin was assessed as an infection imaging agent in a rat model by comparison with (99m)Tc-DTPA and (99m)Tc-leukocytes. (99m)Tc-alafosfalin was prepared via an instant cold kit and (99m)Tc-leukocytes were prepared using (99m)Tc-stannous fluoride colloid in an ex vivo labeling procedure of whole blood. In separate experiments, the three radiotracers were administered to rats infected with staphylococcus aureus. Quantitative biodistribution studies were performed as well as scintigraphic images and histopathology. (99m)Tc-alafosfalin is a stable product, obtained in high radiochemical purity (>95%). This agent was mainly renally excreted, with low liver, spleen and bone uptake, and resulted in a mean ratio of infected/non-infected thighs of 4.3/1.0 at 4 hr post radiotracer injection. (99m)Tc-DTPA gave a corresponding ratio of 1.9/1.0 and (99m)Tc-leukocytes gave 20.0/1.0 at the same time point. An in vitro assay found the level of (99m)Tc-alafosfalin binding to staphylococcus aureas higher than (99m)Tc-DTPA (10% versus 1% respectively). (99m)Tc-alafosfalin accumulates at sites of infection in a rat model better than the perfusion molecule (99m)Tc-DTPA, yet less than (99m)Tc-leukocytes. The distribution characteristics of this (99m)Tc-antibiotic peptide would be an advantage in imaging abdominal and soft tissue infection.