Zn 2+ responsive MRI contrast agents are typically composed of a Gd-chelate conjugated to a Zn 2+ binding moiety via a linker, and allow for Zn 2+ detection in the presence of Human Serum Albumin (HSA). In order to decipher key parameters driving their Zn 2+-dependent MRI response, we designed a pyridine-based ligand, PyAmC2mDPA, and compared the properties of GdPyAmC2mDPA to those of analogue complexes with varying Gd-core, Znbinding moiety or linker size. Stability constants determined by pH-potentiometry show good selectivity of PyAmC2mDPA for Gd 3+ (Log K Gd = 16.27) vs Zn 2+ (Log K Zn = 13.58), and prove that our modified Zn 2+-binding DPA moiety prevents the formation of previously observed dimeric species. Paramagnetic Relaxation Enhancement measurements indicate at least three sites available for GdPyAmC2mDPA binding on the HSA, and a twofold affinity increase when Zn 2+ is present (K D = 170 µM vs K DZn = 60 µM). Fluorescence competition experiments evidence the higher affinity for site II and the importance of both the Zn-binding part and the Gd-core to generate enhanced HSA affinity in the presence of Zn 2+. Finally, the analysis of Nuclear Magnetic Relaxation Dispersion (NMRD) data suggests significantly increased rigidity for the Zn 2+-bound system which is responsible for the Zn 2+-dependent relaxivity response.