The antibiotic Zeocin, a derivative of phleomycin, was evaluated for use as a selection system in both dipteran and lepidopteran insect cell lines. Growth of Drosophila cell lines, Kc1 and SL2, was inhibited at Zeocin concentrations of 50 and 75 microg/ml, respectively, while the Spodoptera cell line, Sf9, was inhibited at a concentration of 250 microg/ml Zeocin. The mammalian cytomegalovirus (CMV) and Simian virus 40 (SV40) early promoters did not function in these insect cell lines. Several baculovirus-derived immediate-early (IE) promoters from the Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV) and Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) were used to drive expression of the Zeocin resistance gene (ble) in these cell lines. The resulting plasmid vectors enabled selection of Zeocin-resistant cell lines within 3-4 weeks. Gene amplification events in the presence of increasing Zeocin concentrations were not detected using Southern blot analysis. Furthermore, the function of the baculovirus IE promoters, as demonstrated by beta-galactosidase expression, was not detectable in a variety of mammalian cell lines tested. A cloning/shuttle vector, containing ten unique restriction sites, was constructed which allows for selection of Zeocin resistance in insect cell lines and in Escherichia coli.