The avidin-biotin system is a fundamental technology in biomedicine for immunolocalisation, imaging, nucleic acid blotting and protein labelling. This technology has recently been adapted for use in gene therapy vector applications to add proteins or cell-targeting ligands to non-viral and viral vectors. Two biotinylation technologies are being used in these applications: chemical biotinylation and metabolic biotinylation. In chemical biotinylation, reactive alkylating agents couple biotin to proteins by random covalent attachment to amino acid side chains. In metabolic biotinylation, proteins are genetically engineered with a biotin acceptor peptide (BAP), such that they are covalently biotinylated by cellular biotin ligases during viral vector production. Both technologies show promise for cell-targeting in vitro and in vivo, and for ligand screening applications. Metabolic biotinylation has the added feature of allowing viruses, vectors and vaccines to be produced from cells already biotinylated, thereby allowing them to purified by affinity chromatography on monomeric avidin columns.