Ternary ligand technetium complexes of a hydrazinonicotinamide-conjugated cyclic peptide (HYNICtide: cyclo(D-Val-NMeArg-Gly-Asp-Mamb(5-(6-(6-hydrazinonicotinamido)hexanamide)))) and 2-hydrazinopyridine (HYPY) were prepared and characterized by various spectroscopic methods. The HPLC concordance experiments for (99m)Tc and (99)Tc analogues show clearly that the same complexes are prepared on the no-carrier-added ((99m)Tc) and the carrier-added ((99)Tc) levels. Using a chirality experiment, it was demonstrated that the presence of two radiometric peaks in the HPLC chromatograms of RP444, RP445, and RP446 is due to the resolution of diastereomers, which result from the presence of chiral cyclic peptide and the formation of two enantiomers of the technetium chelate. In a ligand challenge experiment, we found that the high solution stability of these ternary ligand [(99m)Tc]HYNICtide complexes is due to their kinetic inertness. The 1:1:1:1 composition for Tc:HYNICtide:L:tricine (L = TPPTS, TPPDS, and TPPMS) in these ternary ligand [(99)Tc]HYNICtide complexes is confirmed by (1)H NMR and FAB mass spectral data and is completely consistent with that determined on the tracer ((99m)Tc) level. In addition, the IC(50) values of RP444, RP445, and RP446 and the two isomeric forms of RP444 were determined using a platelet IIb/IIIa binding assay. Both isomeric forms of RP444 were found to have the same binding affinity (IC(50) = 13 +/- 2 nM). Complexes [(99)Tc(HYPY)(PPh(3))(2)Cl(2)] and [(99)Tc(HYPY)(PPh(3))(tricine)] were isolated from the reaction of HYPY with [n-Bu(4)N][TcOCl(4)(-)] in the presence of excess tricine and triphenylphosphine. [(99)Tc(HYPY)(PPh(3))(tricine)] serves as a model for ternary ligand [(99m)Tc]HYNICtide complexes. Both complexes have been characterized by HPLC, spectroscopic (IR, NMR, and FAB-MS) methods, and elemental analysis. The HPLC concordance for complexes [(99m)Tc(HYPY)(PPh(3))(tricine)] and [(99)Tc(HYPY)(PPh(3))(tricine)] shows that the two complexes are identical. The NMR ((1)H and (13)C) data suggests that the complex [(99)Tc(HYPY)(PPh(3))(tricine)] have an octahedral coordination geometry with a monodentate diazenido HYPY, a tetradentate tricine, and a monodentate triphenylphosphine coligand.