A new molecular metallic fragment for labeling biologically active molecules with 99mTc and 188Re is described. This system is composed of a combination of tridentate π-donor and monodentate π-acceptor ligands bound to a [M Ξ N]2+ group (M = (99m)Tc, 188Re) in a pseudo square-pyramidal geometry. A simple structural model of the new metallic fragment was obtained by reacting the ligand 2, 2'-iminodiethanethiol [H2NS2 = NH(CH2CH2SH)2] and monodentate tertiary phosphines with the [M Ξ N]2+ group (M = (99m)Tc, (188)Re). In the resulting complexes (dubbed3+1complexes), the tridentate ligand binds the [M Ξ N]2+ core through the two deprotonated, negatively charged, thiol sulfur atoms and the neutral, protonated, amine nitrogen atom. The residual fourth position of the five-coordinated arrangement is occupied by a phosphine ligand. The chemical identity of these model (99m)Tc and (188)Re compounds was established by comparison with the chromatographic properties of the corresponding complexes obtained at the macroscopic level with the long-lived (99)Tc and natural Re isotopes. The investigation was further extended to comprise a series of ligands formed by simple combinations of two basic amino acids or pseudo-amino acids to yield potential tridentate chelating systems having [S, N, S] and [N, N, S] as sets of π-donor atoms. Labeling yields and in vitro stability were investigated using different ancillary ligands. Results showed that SNS-type ligands afforded the highest labeling yields and the most robust 3+1 nitrido complexes with both (99m)Tc and (188)Re. Thus, the new chelating system can be conveniently employed for labeling peptides and other biomolecules with the [M Ξ N]2+ group.