Complex binding of proteins by metal-chelating Lipids via surface-exposed or protein-engineered histidines provides an universal and powerful concept for the orientation and two-dimensional crystallization of proteins at self-organized interfaces. To demonstrate pair formation between individual histidine-tagged molecules and chelator lipids on the molecular level, we have synthesized novel lipids bearing both a Ni-NTA chelator and a fluorescent group. These lipids serve as spectroscopic probes to visualize directly the molecular recognition of fluorescence-labeled histidine-tagged peptides by metal-chelating lipids using fluorescence resonance energy transfer (FRET). The molecular docking to chelator lipids assembled in mono- or bilayers is highly specific, revealing only 3% unspecific adsorption and a binding constant of 3 mu M. The affinity constant was confirmed by fluorescence correlation spectroscopy (FCS) on single molecules, where the ratio of lipid-bound to free was analyzed by their intrinsically different diffusion times passing through a confocal volume of about 1 fL. By using a model peptide most of the electrostatic and steric contribution to the binding process can be neglected. Therefore, the affinity constant can serve as a standard value for the binding of histidine-tagged proteins to chelator lipid interfaces.