Equine infectious anemia virus (EIAV) is a lentivirus in the retrovirus family of viruses. Replication-defective EIAV vectors have been constructed that encode bacterial puromycin-N-acetyl transferase and E. coli β-galactosidase. These vectors could be prepared with titers greater than 105 infectious units/ml and were able to act as vehicles to carry genes into cultured human cells. In addition, stable helper cell lines were created by modifying human 293 cells to express EIAV proteins. Unlike retroviral vectors based on murine leukemia virus, EIAV lentiviral vectors transduce nondividing (aphidicolin-arrested) cells. These properties make EIAV vectors promising gene transfer vehicles.