In our previous studies, interleukin-8 (IL-8) was labeled with (99m)Tc using hydrazinonicotinamide (HYNIC) as bifunctional coupling agent and tricine as coligand. This preparation showed excellent characteristics for imaging of infection in a rabbit model of soft-tissue infection. In the present study, the propylaldehyde hydrazone formulation of HYNIC was introduced to stabilize HYNIC-IL-8. (99m)Tc-HYNIC-IL-8 was prepared using 5 different coligand formulations. The effect of these coligand formulations on the in vitro characteristics and in vivo behavior of (99m)Tc-HYNIC-IL-8 was investigated. HYNIC-conjugated IL-8 was labeled with (99m)Tc in the presence of either (A) tricine, (B) ethylenediaminediacetic acid (EDDA), (C) tricine and trisodium triphenylphosphinetrisulfonate (TPPTS), (D) tricine and nicotinic acid (NIC), or (E) tricine and isonicotinic acid (ISONIC). These preparations were characterized in vitro by RP-HPLC, determination of the octanol/water partition coefficient, stability studies, and receptor binding assays. The in vivo biodistribution of the radiolabel in rabbits with E. coli-induced soft-tissue infection was determined both by gamma-camera imaging as well as by tissue counting at 6 h pi. Specific activity (MBq/microg) was highest for (ISO)NIC (up to 80) > TPPTS (40) > tricine (15) > EDDA (7). RP-HPLC and octanol/water partition coefficients showed a shift toward higher lipophilicity for the TPPTS preparation. The leukocyte receptor binding fractions were around 40-55% for all preparations except for TPPTS, which showed predominantly nonspecific binding. All preparations were stabilized in serum, but the stability in PBS was highest for NIC and TPPTS > EDDA > ISONIC > tricine. The in vivo biodistribution showed highest abscess/muscle for NIC and ISONIC (>200) > EDDA and tricine (approximately 100) > TPPTS (<40). Gamma camera imaging rapidly visualized the abscess from 2 h pi onward for all formulations. The abscess/background (A/B) at 6 h pi for ISONIC was significantly higher (P < 0.05) than that of tricine and the A/B of TPPTS was significantly lower (P < 0.05). IL-8 can be rapidly and easily labeled with (99m)Tc using HYNIC as a chelator in combination with various coligands. The most optimal infection imaging characteristics were found for formulations using nicotinic acid/tricine as coligand system combining a high specific activity and high in vitro stability with high abscess/muscle ratios (>200) and high abscess/background ratios (>20). Protein doses to be administered were as low as 70 ng/kg bodyweight. At these low protein doses, side effects are not to be expected in the human system. This paves the way for infection imaging studies in patients.