Retroviruses are used for a variety of applications requiring the delivery of exogenous genes to cells and animals. For many of these applications, including gene therapy, safer and more efficient retroviral vectors are needed. Vectors based on Harvey murine sarcoma virus (HaMSV) are attractive because nearly all their viral sequences outside of the LTRs are derived from rat endogenous VL30 retroviruses. These sequences are not homologous to the functional viral mRNAs in commonly used retrovirus packaging cell lines, the packaging and dimerization domains of HaMSV are small and contain no splice donor sites, and the 5' sequences of HaMSV appear to confer efficient packaging and stability on genomic RNAs. HaMSV/MDR1 vectors use the human multidrug resistance gene as a dominant, selectable, amplifiable marker for gene delivery, but current versions of these vectors are large, with over 3300 nt of HaMSV sequences downstream of MDR1. We analyzed the requirement for these downstream sequences in HaMSV vectors and found that modified HaMSV/MDR1 vectors lacking virtually all viral sequences downstream of MDR1 support the production of high-titer retroviruses and the efficient transduction, selection, and amplification of MDR1. A reduced-size HaMSV/MDR1 vector was further modified to include a second heterologous gene under the control of an internal SV40 promoter. Using MDR1 as a selectable marker, we obtained efficient virus production, gene transduction, and expression of MDR1 plus the heterologous gene.