Site-specific, enzymatic biotinylation of recombinant proteins can be exploited to circumvent many problems associated with the use of biotinylating reagents in vitro and to overcome some of their inherent limitations. Additionally, biotinyl proteins can be purified to near-homogeneity in a single step under native conditions. Here we report that a biotin acceptor peptide (BAP) substrate for Escherichia coli biotin holoenzyme synthetase (BirA) can be used to label recombinant proteins with biotin in Spodoptera frugiperda (Sf9) cells, and we describe a collection of baculovirus transfer vectors specifically designed for this purpose. These BioBac vectors will greatly expand the range of proteins to which this technology can be applied.