This study represents a first comprehensive investigation on how the decorporation agents CaNa3-DTPA (DTPA) and 3,4,3-LI(1,2-HOPO) (LIHOPO) affect EuIII interactions with human and rat kidney cells in vitro. Cell biological investigations were complemented with physicochemical measurements to correlate cytotoxic impairments with intracellular metal uptake and EuIII speciation. Upon exposure to sole DTPA or LIHOPO, cell viability and morphology are affected in a time- and concentration-dependent manner. For both decorporation agents, detailed EC50 values for renal cells in vitro are reported. Simultaneous application of EuIII + DTPA in the medium leads to formation of the soluble and largely cell impermeable EuDTPA2- complex. At ligand excess, this significantly reduces intracellular EuIII uptake. However, EuDTPA2- was spectroscopically detected also inside cells indicating that small fractions of this complex are able to pass the plasma membrane. When EuIII + LIHOPO is applied to the medium, the soluble EuLIHOPO- complex is formed. In contrast to DTPA, this drastically enhances intracellular EuIII uptake even at ligand deficit demonstrating that EuLIHOPO- is highly cell permeable. Concomitantly, this complex was spectroscopically detected inside cells confirming its plasma membrane passage and intracellular stability. Nevertheless, due to stable EuIII binding, the cell viability is not influenced by the increased intracellular EuIII content. In fact, the applied ligand concentration is much more critical in this regard, emphasizing the need for cytotoxic investigations. Our results improve the knowledge of the cellular interactions of lanthanides ± decorporation agents and demonstrate the combination of in vitro cell culture and spectroscopy being a sophisticated toolbox for this.