EC20, a folate-targeted (99m)Tc based radioimaging agent with a high folate receptor (FR) binding affinity, has been used for both the diagnosis and the staging of FR positive malignancies (currently in phase III trials) and also for the localization of inflamed lesions characterized by the accumulation of FR+ macrophages. Because recent evidence has suggested that FR+ macrophages might accumulate at sites of infectious disease, this study evaluated whether EC20 might prove similarly useful for imaging bacterial infection foci. Using gamma scintigraphic imaging, it was demonstrated that EC20 accumulated at sites of Staphylococcus aureus infection with a significant difference (P < 0.0001, n = 12) in enrichment noted between infected and noninfected limbs. Confirmation that the elevated uptake of EC20 in infected limbs was FR-mediated was supported by suppression of EC20 accumulation in the presence of a 200-fold excess of free folic acid (P < 0.0001, n = 12). This study establishes for the first time the use of EC20 to image and localize sites of infectious disease.