Recombinant rabies virus (RV) vaccine strain-based vectors have been successfully developed as vaccines against other viral diseases (J. P. McGettigan et al., J. Virol. 75:4430-4434, 2001; McGettigan et al., J. Virol. 75:8724-8732, 2001; C. A. Siler et al., Virology 292:24-34, 2002), and safety concerns have recently been addressed (McGettigan et al., J. Virol. 77:237-244, 2003). However, size limitations of the vectors may restrict their use for development of vaccine applications that require the expression of large and multiple foreign antigens. Here we describe a new RV-based vaccine vehicle expressing 4.4 kb of the human immunodeficiency virus type 1 (HIV-1) Gag-Pol precursor Pr160. Our results indicate that Pr160 is expressed and processed, as demonstrated by immunostaining and Western blotting. Electron microscopy studies showed both immature and mature HIV-1 virus-like particles (VLPs), indicating that the expressed HIV-1 Gag Pr55 precursor was processed properly by the HIV-1 protease. A functional assay also confirmed the cleavage and functional expression of the HIV-1 reverse transcriptase (RT) from the modified RV genome. In the next step, we constructed and recovered a new RV vaccine strain-based vector expressing a chimeric HIV-1(89.6P) RV envelope protein from an additional RV transcription unit located between the RV nucleoprotein (N) and phosphoprotein (P) in addition to HIV-1 Pr160. The 2.2-kb chimeric HIV-1/RV envelope protein is composed of the HIV-1 Env ectodomain (ED) and transmembrane domain (TD) fused to RV glycoprotein (G) cytoplasmic domain (CD), which is required for efficient incorporation of HIV-1 Env into RV particles. Of note, the expression of both HIV-1 Env and HIV-1 Pr160 resulted in an increase in the rhabdoviral genome of >55%. Both rhabdovirus-expressed HIV-1 precursor proteins were functional, as indicated by RT activity and Env-based fusion assays. These findings demonstrate that both multiple and very large foreign genes can be effectively expressed by RV-based vectors. This research opens up the possibility for the further improvement of rhabdovirus-based HIV-1 vaccines and their use to express large foreign proteins, perhaps from multiple human pathogens.