The development of transfection procedures that optimize the efficiency of recombination events is central to the isolation of recombinant baculoviruses for foreign gene expression. A variety of transfection methods, which were first developed for mammalian cell lines, have been adapted for use with insect tissue-culture cells. Conditions required for the maximum transfection efficiency of any vertebrate or invertebrate cell line vary considerably. This chapter will focus on the methods and conditions that have been employed with lepidopteran tissue-culture cells. In general, these conditions have been developed for efficient transfection of Bombyx mori (Bm-N), Spodopteru frugiperda (IPLB-SF-21-AE), and SF9 (a subclone of IPLB-SF-21-AE) cell lines. However, we have also successfully used these procedures for transfection of Lymantria dispar (IPLB-LD-652Y) and Trichoplusia ni (TN-368) tissue-culture cells. The medium in which lepidopteran cells are grown, e.g., TNMFH, TC-100 or serum-free media, may influence the transfection efficiency. For the isolation of baculovirus expression vectors, cotransfection is generally performed using viral DNA and a bacterial plasmid DNA that functions as a transfer vector. The transfer vector contains a foreign gene inserted downstream from a baculovirus gene promoter and a sufficient amount of viral flanking DNA sequences to facilitate recombination between the plasmid and viral DNA sequences (allelic transplacement). Most research has been conducted using allelic transplacement of the polyhedrin gene with foreign gene inserts.