In an effort to devise simple and robust systems that can reproduce in synthetic membranes important features of biological targeting and surface assembly, a versatile system for targeting proteins to lipid membranes has been developed. This system utilizes metal-chelating iminodiacetate (IDA) lipids loaded with divalent metal ions (Cu2+ or Ni2+) to target proteins genetically modified with a poly(histidine) fusion peptide. The new pyrene-labeled iminodiacetate lipid 2 can be used for fluorescence imaging and spectroscopic studies of lipid reorganization induced by protein binding and assembly on lipid membranes. Metal-chelating IDA lipids 1 and 2 target the soluble domain of cytochrome b(5) to lipid assemblies by sharing the metal ion with a six-histidine sequence appended to the protein C-terminus. Protein binding to Langmuir monolayers containing the IDA-Cu2+ lipids 1 and 2 is observed by monitoring increases in the monolayer area at a surface pressure high enough to block nonspecific protein insertion (25 mN/m). The His-tagged cytochrome b(5) binds the Cu2+-loaded 2 monolayer with high affinity (K-d < 50 nM). No binding is observed in the absence of metal ions or for cytochrome b(5) without the 6-His fusion peptide. Specific protein targeting to the monolayer loaded with Ni2+ is confirmed by fluorescence microscopy of fluorescein-labeled 6-His cytochrome b(5). The poly(histidine) fusion peptide, widely used for recombinant protein purification, makes this targeting approach applicable to a large number of proteins.