Using a complementation assay, we have evaluated the potential of two eukaryotic expression systems to produce functional virus proteins. The first expression system was based on a bovine papilloma virus (BPV) eukaryotic expression vector which contained a copy of the gene for the membrane glycoprotein G of vesicular stomatitis virus (VSV). This vector was transfected into a mouse cell line, and transformed cell clones constitutively expressing VSV G protein were selected. These cell clones were then screened for their ability to support the replication of a temperature-sensitive G mutant of VSV (ts045) at the permissive and nonpermissive temperatures. A 100-fold increase in ts045 titer was observed in some of the G protein-producing cell lines in comparison with nonproducing cells. These results were compared with complementation by VSV G protein expressed from a second expression system utilizing a vaccinia virus (VV) recombinant which produced bacteriophage T7 RNA polymerase. T7 RNA polymerase expressed in cells infected with the vaccinia recombinant produced VSV G transcripts from a plasmid which had been transfected into these cells. This plasmid contained the VSV G gene cloned between T7 RNA polymerase initiation and termination signals. VSV G protein expressed by this system was able to complement ts045 replication at the nonpermissive temperature, and yielded much greater levels of complemented virus than the BPV system. When calcium phosphate-mediated transfection was used to introduce the VSV G plasmid vector into cells infected with the VV recombinant, a complementation efficiency as high as 1500-fold was obtained. Using lipofectin-mediated transfection, a 15,000-fold increase in virus titer could be obtained in G protein-producing cells in contrast to nonproducing cells. At the nonpermissive temperature, yields of temperature-sensitive virus were within 10-fold of the yields obtained at the permissive temperature. Virus produced in this system was shown to be a pseudotype which contained wild-type G protein in the viral envelope but still maintained the temperature-sensitive genotype. This expression system will be used to study the extent to which the integrity of the G coding sequence of wild-type VSV might be altered in the absence of selection pressure for functional G protein during VSV replication. © 1990.