The baculovirus/insect cell heterologous expression system provides an important tool for investigating the catalytic activity of individual drug-metabolizing enzymes toward a particular substrate. In this chapter we describe a baculovirus/insect cell system that we have used for the expression of human and mouse flavin-containing monooxygenases. Methods are described for the generation of recombinant baculoviral DNAs, via both site-specific transposition in Escherichia coli and site-specific recombination in vitro; adaptation of Spodoptera frugiperda (Sf ) 9 cells to shaking culture and to serum-free medium; cryopreservation and transfection of Sf 9 cells; amplification of baculovirus and determination of viral titer; analysis of baculoviral DNA; and expression and analysis of recombinant proteins.