We used a well established transient expression assay to test the ability of the baculovirus Spodoptera exigua M nucleopolyhedrovirus (SeMNPV) homologs of Autographa californica MNPV (AcMNPV) late expression factors (lefs) to activate a late promoter-reporter gene cassette in SF-21 cells. This insect-derived cell line is fully permissive for AcMNPV infection but not for SeMNPV. In the assay, 19 AcMNPV lefs stimulate optimal levels of late gene promoter activity. SeMNPV lef-5 successfully replaced the corresponding AcMNPV gene in the context of the remaining set of AcMNPV lefs, whereas SeMNPV dnapol and 39k exhibited partial activity. When all the SeMNPV lefs were assayed together or in the presence of four lefs encoded only in AcMNPV, it resulted in background levels of late promoter-driven reporter gene activity. However, SeMNPV genomic DNA and the four AcMNPV-specific lefs stimulated low levels of reporter gene activity. Moreover, SeMNPV IE-1, but not AcMNPV IE-1, further stimulated late gene expression in the presence of SeMNPV DNA. AcMNPV IE-1 was able to mediate early gene expression cis-linked to homologous regions (hrs) derived from AcMNPV and SeMNPV. In contrast, SeMNPV IE-1 was more specific for SeMNPV-derived hr elements.