An amphotropic retrovirus packaging cell line was constructed in which the gag, pol, and env genes of the helper virus are separated on two different plasmids and in which the packaging signals and 3' long terminal repeats are removed. To do this, a plasmid containing the Moloney murine leukemia virus gag and pol gene was transfected into NIH 3T3 cells, and a plasmid containing the 4070A amphotropic env gene was transfected into one of the resulting clones which produced a high level of reverse transcriptase. A clone producing a high level of amphotropic env protein (GP + envAm12) was then isolated. When transfected into GP + envAm12 cells, titers of the retroviral vector N2, containing a neomycin resistance gene, ranged from 10(2) to greater than 10(6) CFU/ml on 3T3 cells, from 1.3 x 10(4) to 2.7 x 10(5) CFU/ml on HeLa cells, and from 1.0 x 10(2) to 6.0 x 10(3) CFU/ml on K562 cells when assayed by G418 resistance. These titers were comparable to titers obtained using the PA317 cell line. Tests for the safety of the GP + envAm12 packaging line showed no evidence for the generation of wild-type virus. Thus, the efficiency and safety of the GP + envAm12 cell line in gene transfer into human cells may provide an optimal system for experiments whose goal is human gene therapy.