The ability of 12 unique lepidopteran insect cell lines from Anticarsia gemmatalis, Heliothis virescens, Lymantria dispar (two lines), Mamestra brassica, Plutella xylostella, Spodoptera frugiperda (two lines), and Trichoplusia ni (three lines) to support production of a recombinant polydnavirus (PDV) protein (GiPDV 1.1) expressed using the Bac-to-Bac™ baculovirus expression system was examined. Polydnavirus gene GiPDV 1.1 was cloned into the pFastBac baculovirus vector under the control of the polyhedron promoter, followed by generation of recombinant bacmid–GiPDV 1.1 by site-specific transposition. The ability of each insect cell line to support recombinant PDV gene expression was estimated using reverse transcriptase–polymerase chain reaction and Western blot. Each insect cell line infected with recombinant bacmid–GiPDV 1.1 and tested in this study was capable of supporting and producing recombinant protein. Time course expression analysis showed that 72–96 h after transfection to be the optimal time for harvest of recombinant protein for each insect cell line.