Rationale and Objectives: Four new Gd-DTPA derivatives substituted by hydrophobic alkyl chains have been synthesized and characterized: Gd-DTPA-Bdodecylamide 1, Gd-DTPA-Bcarboxylundecylamide 2, Gd-C 4 -dodecylisothiourea-Bz-DTPA 3 and Gd-C 4 -carboxylundecyl isothiourea-Bz-DTPA 4 (Fig. 1). Their affinities for HSA were evaluated by two techniques: proton relaxation rate and electrospray mass spectrometry. Methods: Gd complexes were synthesized by a described method (1,2). NMRD profiles were obtained at 310 K on a field cycling relaxometer (Stelar, Italy). Additional longitudinal relaxation rates were measured on Bruker Minispecs at 20 and 60 MHz. Electrospray mass spectra were obtained on a Q-TOF 2 (Micromass, UK). Results: The NMRD profile in water of bisamide 1 shows the spontaneous formation of micelles in solution whereas those of the three other complexes have an evolution characteristic of small Gd complexes. In the presence of HSA, complexes 1-3 show a weak increase of their relaxation rates that demonstrates low affinity for the protein. For compound 4, there is a clear hump at high fields comparable to the one reported in the literature for strong HSA binders like MS-325. Additionnally, the stability vs zinc transmetallation followed by proton relaxometry is significantly higher for the C 4 derivatives (3 and 4) than for the bisamide structures (1 and 2). The binding of complex 4 to HSA was further investigated through data characterizing the evolution of the proton paramagnetic relaxation rate of a 4% HSA solution containing increasing amounts of the gadolinium complex. The fitting of the titration curves gave an association constant of about 9410 AE 2560 M À1 with 2.0 AE 0.2 binding sites and an r 1 c of 43.0 AE 2.4 s À1 mM À1 . The electrospray mass spectrometry confirms the strong interaction and shows a signal corresponding to a complex between the protein and one molecule of contrast agent. Conclusions: These results confirm the benefit of grafting the lipophilic substituents on the C 4 position of the backbone and of the presence of a negative charge on this chain, which seems essential to the interaction with HSA.