The aim of this study was to develop ^99mTc(CO)₃-labeled fluoroquinolones as novel SPECT radiopharmaceuticals for imaging bacterial infection. Fluoroquinolones, e.g., ofloxacin (OFX), levofloxacin (LVX), lomefloxacin (LMX) and norfloxacin (NFX) were labeled with a fac-[^99mTc(CO)₃(H₂O)₃]⁺ precursor. The radiochemical purity of the radiopharmaceuticals exceeded 97% as determined by thin layer chromatography and HPLC. No further purification was necessary before injection. The Re(CO)₃ complex of one of the fluoroquinolones (levofloxacin) was synthesized using [Re(CO)₃(H₂O)₃]OTf and Re(CO)₅Br precursors in separate experiments and characterized by IR, NMR and mass spectroscopic analysis. These studies revealed the formation of a single species in which the piperazinyl nitrogen and the –COOH group attached to the benzoxazine ring system of quinolone were involved in co-ordination to the Re(CO)₃ core. The HPLC elution pattern and retention time of the Re(CO)₃-LVX complex were comparable to those of the corresponding ^99mTc(CO)₃-complex proving their similarity. When incubated in isotonic saline and serum up to 24 h ^99mTc(CO)₃-labeled fluoroquinolones exhibited good in vitro stability. Biodistribution studies performed at different time points on rats intramuscularly infected with S. aureus as well as on rats with sterile inflammation revealed a higher uptake in the infected area than the turpentine induced inflamed area. The uptake in infected thigh was significant with ^99mTc(CO)₃-OFX followed by ^99mTc(CO)₃-LVX. The mean ratios of the uptake in infected/non-infected thighs were 4.75 and 4.27 at 8 h and 24 h, respectively, for ^99mTc(CO)₃-OFX and 4.42 and 4.18 at 24 h and 8 h, respectively, for ^99mTc(CO)₃-LVX. The above abscess to muscle ratios were higher than reported for ^99mTc-ciprofloxacin and other ^99mTc-labeled fluoroquinolones. Scintigraphy studies also showed a significant uptake in the infectious lesions suggesting that ^99mTc(CO)₃-fluoroquinolones might be useful as diagnostic agents for targeted delivery in bacterial infection.