The enzyme BirA is a key reagent because of its ability to biotinylate proteins at a specific residue in a recognition sequence. We have used this enzyme to biotinylate the C termini of membrane proteins, allowing these proteins to be tetramerized by binding to streptavidin. Because of the specificity of the biotinylation at the C terminus, the orientation of the membrane proteins on the streptavidin is equivalent to that of the native protein on the cell surface. These tetrameric proteins can be used to study protein receptor-ligand interactions at the cell surface, and site-specific biotinylation can be used to study proteins in vitro using a defined orientation. The biotinylation of histones by BirA ligase is consistent with the proposed role of human HCS in chromatin. The N-terminal BirA domain is required for both transcriptional regulation of biotin synthesis and biotin protein ligase activity.