Hydrazinonicotinamide (HYNIC) derivatized chemotactic peptides radiolabeled with 99mTc- (via 99mTc-glucoheptonate) have been demonstrated to be useful for infection imaging [J. Nucl. Med. 34, 1964-1974 (1993)]. Since HYNIC can occupy only two sites of the technetium co-ordination sphere, the labeled product most probably contains additional ligands. Thus we hypothesized that glucoheptonate serves this role by acting as a "'co-ligand'". Due to the low molecular weight of the chemotactic peptides, the "co-ligand" used for technetium labeling could have profound effects on biodistribution. To evaluate this possibility, we measured the biodistribution of 99mTc-labeled For-MLFK-HYNIC radiolabeled using four different "co-ligand"s: glucarate, glucoheptonate, mannitol and glucamine, providing a small series of hydroxyl-backbone ligands which differ in the number and type of ionizable functional groups present. Each preparation was injected into groups of 6 rats (approximately 10 microCi/rat) and biodistribution was determined at 5, 30, 60 and 120 min. Although small differences in biodistribution were detected in most tissues, the most prominent differences (P < 0.01) were observed in lung (glucoheptonate, glucarate > mannitol >> glucamine), liver (glucarate, glucoheptonate, mannitol >> glucamine), kidney (mannitol > glucarate, glucoheptonate, glucamine), spleen (glucarate >> glucoheptonate, mannitol >> glucamine) and GI-tract (glucarate, glucamine >> gluco-heptonate >> mannitol). These results provide support for the "co-ligand" hypothesis and indicate that the nature of the "co-ligand" can have profound effects on biodistribution. Although radiolabeling using glucamine as the "co-ligand" results in the lowest concentrations of radioactivity in most organs, the extremely low concentration of mannitol-labeled peptide in the GI-tract suggests that this may be the "co-ligand" of choice for most applications.