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 Tc-99m and Tc-99 analogues show clearly that the same complexes are prepared on the no-carrier-added (Tc-99m) and the carrier-added (Tc-99) 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 [Tc-99m]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 [Tc-99]HYNICtide complexes is confirmed by H-1 NMR and FAB mass spectral data and is completely consistent: with that determined on the tracer (Tc-99m) level. In addition, the IC50 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 (IC50 = 13 +/- 2 nM). Complexes [Tc-99(HYPY)(PPh3)(2)Cl-2] and [Tc-99(HYPY)(PPh3)(tricine)] were isolated from the reaction of HYPY with [n-Bu4N]TcOCl4-] in the presence of excess tricine and triphenylphospbine. [Tc-99(HYPY)(PPh3)(tricine)] serves as a model for ternary ligand [Tc-99m]HYNICtide complexes. Both complexes have been characterized by HPLC, spectroscopic (IR, NMR, and FAB-MS) methods, and elemental analysis. The HPLC concordance for complexes [Tc-99m(HYPY)(PPh3)(tricine)] and [Tc-99(HYPY)(PPh3)(tricine)] shows that the two complexes are identical. The NMR (H-1 and C-13) data suggests that the complex [Tc-99(HYPY)(PPh3)(tricine)] have an octahedral coordination geometry with a monodentate diazenido HYPY, a tetradentate tricine, and a monodentate triphenylphosphine coligand.