Five recombinant baculoviruses, each containing a different insect luciferase gene encoding a protein with characteristic light emission properties, namely, luc GR (546 nm), luc FF (556 nm), luc YG (560 nm), luc YE (578 nm), and luc OR (593 nm) were constructed. All genes were inserted under the transcriptional control of the polyhedrin gene promoter of the Autographa californica nuclear polyhedrosis virus (AcNPV) and expressed in Spodoptera frugiperda insect cells during viral infection. The biological activity of the different luciferases was characterized by using intact recombinant baculovirus infected cells. Addition of the substrate, D-luciferin, immediately prior to the analysis allowed monitoring of light emission by flow cytometry. Also, the kinetics of the light emission of lucGR was analyzed with the flow cytometer. The emission peaks of the infected cells were clearly separated by wavelength scanning. Especially, the firefly luciferase (lucFF) had a broad peak and transient luminescence. The highest maximal intensity values in vivo were recorded for luc GR and luc YG. SDS-PAGE analysis showed that the major protein expressed had a molecular weight similar to authentic luciferase. Flow cytometry and insect luciferases with clearly separated emission spectra appear to be of value for sensitive in vivo analysis of gene promoter activity.