A lentivirus-based packaging system was designed to reduce the chance of recombination between helper and gene transfer vector sequences by using the constitutive transport element (CTE) derived from Mason-Pfizer monkey virus for expression of the viral proteins and the Rev-Rev response element (RRE) combination for expression of the gene transfer vector. Using this approach, we evaluated a series of human immunodeficiency virus type 1 packaging constructs that express one or more accessory proteins (Vif, Vpr, and Vpu), in addition to the Gag and Pol proteins, for particle formation and virus stock production for gene transfer. Constructs that also express Vpr or both Vpr and Vpu produced more particles, as measured by a p24 assay, than did plasmids that did not contain these sequences. Transactivation experiments showed that the packaging plasmids that encode Vpr or both Vpr and Vpu also expressed a functional single-exon Tat protein. For these constructs, high-titer virus stocks could be prepared in the absence of a cotransfected Tat-expressing plasmid. Amphotropic-envelope-pseudotyped virus stocks prepared with all of the packaging constructs, irrespective of whether any of the accessory proteins were coexpressed, were equally efficient in transducing growth-arrested HeLa cells. The combination/mixed packaging system was compared to systems that were based on either the CTE alone or Rev and RRE for expression of both the packaging plasmid as well as the gene transfer vector. The combination/mixed packaging system was comparable to the other systems for production of virus stocks, suggesting that this design may prove to be safer for the eventual deployment of lentivirus vectors for therapeutic purposes.