Although chronic low-level exposure to Hg and Cd causes human nephrotoxicity, the bioinorganic processes that deliver them to their target organs are poorly understood. Since the plasma protein human serum albumin (HSA) has distinct binding sites for these metal ions, we wanted to gain insight into these translocation processes and have employed size-exclusion chromatography coupled on-line to an inductively coupled plasma atomic emission spectrometer using phosphate-buffered saline mobile phases. When HSA 'labeled' with Hg and Cd (1:0.1:0.1) using 300 μM of L-methionine was analyzed, the co-elution of a single C, S, Cd, and Hg peak was observed, which implied the intact bis-metalated HSA complex. Since human plasma contains small molecular weight thiols and sulfur-containing metabolites, we analyzed the bis-metalated HSA complex with mobile phases containing 50-200 µM of L-cysteine (Cys), D,L-homocysteine (hCys), or glutathione (GSH), which provided insight into the comparative mobilization of each metal from their respective binding sites on HSA. Interestingly, 50 µM Cys, hCys, or GSH mobilized Hg from its HSA binding site but only partially mobilized Cd from its binding site. Since these findings were obtained at conditions simulating near-physiological conditions of plasma, they provide a feasible explanation for the higher 'mobility' of Hg and its concomitant interaction with mammalian target organs compared to Cd. Furthermore, 50 µM Cys resulted in the co-elution of similar-sized Hg and Cd species, which provides a biomolecular explanation for the nephrotoxicity of Hg and Cd.