Bovine leukemia virus (BLV) and human T-cell leukemia virus type I (HTLV-I) proviral clones were used to construct a new type of retroviral vector. The viral genes encoding the nonstructural, regulatory proteins, Tax and Rex, were replaced with the bacterial neomycin resistance gene controlled by strong constitutive promoters; in pBLV-SVNEO, the SV40 early promoter was used, while in pHTLV-CMVNEO, a cytomegalovirus promoter was used. The BLV vector was normal in all respects except for the regulatory proteins, whereas the HTLV-I vector also had a deletion of envelope gene sequences. Transfection of cells with plasmids containing the recombinant viruses resulted in the constitutive expression of the neo gene; in contrast, viral genes were expressed only when both Tax and Rex were supplied in trans. The release of infectious BLV-SVNEO after transfection of cells with PBLV-SVNEO and Tax/Rex expression plasmids was demonstrated by the transfer of G418-resistance to susceptible cells through the filtered culture medium. Production of infectious HTLV-CMVNEO requires complementation with Tax, Rex, and envelope proteins; this has been achieved by stably transfecting an HTLV-I-producing cell line. A variety of cell lines were found to be susceptible to infection with either BLV-SVNEO or HTLV-CMVNEO. These experiments demonstrate the potential utility of these vectors as gene delivery vehicles. More immediately, they will provide a simple means to quantify virus infectivity prerequisite to analysis of virus receptors and effects of antiviral agents and antisera.