Abstract Introduction TNFR2-Fc and IL-1ra-Fc are recombinant cytokine ligands that target TNF and IL-1. TNFR2-Fc-IL-1ra, a dual-domain agent that incorporates both ligands, allows bifunctional binding of IL-1 receptors and TNF. This study was designed to characterize 99mTc-labeled forms of these ligands, 99mTc-IL-1ra-Fc (IF), 99mTc-TNFR2-Fc (TF), and 99mTc-TNFR2-Fc-IL-1ra (TFI), for inflammation imaging. Methods The cytokine ligands were labeled with 99mTc by a direct approach via 2-iminothiolane (2-IT) reduction at various 2-IT/protein molar ratios. In vivo inflammation targeting studies were carried out in a mouse ear edema model created by topical application of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on the right ear of ICR mice. Results Radiolabeling yields increased with increasing amounts of 2-IT. When the 2-IT/protein ratio reached 1000, the radiolabeling yield was greater than 90% without significant colloid production. TPA-treated ears showed high radioligand uptake, which was clearly detected by SPECT and autoradiographic imaging. The activities (%ID/g) in the inflamed and control ears at 3h after injection were 2.76±0.20 vs. 0.69±0.12 for IF, 5.86±0.40 vs. 2.86±0.61 for TF, and 7.61±0.86 vs. 1.99±0.31 for TFI (P<0.05 vs. controls). TFI showed significantly higher uptake in the inflamed ears compared to TF and IF (P<0.05). Blocking study results indicated specificity of radioligand binding with decreased radioactive uptake in the inflamed ears. Western blotting and ELISA analysis further confirmed a high expression of IL-1β and TNF-α in the inflamed ears. Conclusions 99mTc-labeled cytokine ligands are a promising approach for detecting and understanding the inflammatory process. TFI may be more useful than the single-domain ligands for noninvasive detection of inflammatory sites.