Retroviral vectors were constructed in which the U3 promoter/enhancer of Moloney murine leukemia (Mo-MLV) was replaced by the corresponding region from five related murine retroviruses--AKR murine leukemia virus (AKV), Harvey murine sarcoma virus (HaMSV), myeloproliferative sarcoma virus (MPSV), SL3-3, and the NZB-xenotropic virus (Xeno). In these vectors the chimeric long terminal repeat (chLTR) drives the expression of the chloramphenicol acetyl transferase (CAT) reporter gene that is followed by an internal SV40 virus early region promoter linked to the neomycin phosphotransferase II (NEO) gene. As an initial measure of the relative promoter/enhancer strength of the chLTR vectors, the murine NIH-3T3 cell line and the human JURKAT cell lines were transfected and assayed for CAT reporter activity. Relative to the MoMLV vector, the HaMSV construct was the most active in NIH-3T3 cells whereas the SL3-3 vector displayed the greatest activity in JURKAT cells. Retroviral vector producer cell populations and cell clones were established for each chLTR vector, and all were capable of yielding high vector titers (> 10(5) G418R cfu/ml on NIH-3T3). Supernatant from these cells was used to transduce both mouse and human cell lines and primary cells. In NIH-3T3 cells and two murine fibrosarcoma cell lines, the HaMSV chLTR vector was slightly more active than the MoMLV chLTR vector. In the human HepG2 and HeLa cell lines, the MPSV chLTR vector was the most active. Data from the human JURKAT T-cell line and a T cell line derived from an ADA-deficient severe combined immunodeficiency (SCID) patient demonstrate that the SL3-3 chLTR is the most active in these lymphoid cell lines. The greatest difference in the comparison of the different chLTR vectors was observed in primary human umbilical vein endothelial cells, where the MoMLV vector produced up to 100 times more CAT activity than the SL3-3 vector. These data suggest that the use of specific promoter/enhancer elements may lead to higher levels of gene expression following retroviral-mediated gene transfer into specific cell types and these observations may be useful in the design of human gene therapy experiments.