We have analyzed the relative level of gene expression and viral titer from different types of retroviral vectors used for gene therapy, the LTR-based MFG vector and the internal promoter-containing vectors, LNCX, LNSX and LXSN. The CAT gene was used for comparison of retroviral vector gene expression in both transfected and transduced cells, while the neo gene was used to evaluate viral liter. In transfected cells, MFG-CAT expressed higher levels of CAT then the other vectors, LNC-CAT was next, while L-CAT-SN and LNS-CAT produced much lower levels. CAT expression from MFG-CAT was particularly high in the human T lymphoid cell lines CEM-SS and H9. In nonselected transduced cells. CAT expression from MFG was 10- to 50-fold higher than with the other vectors. Similar observations were made with retroviral constructs expressing human EPO and murine GM-CSF. In transient transfection assays, the titer of MFG was at least five-fold higher than the other vectors as determined by Southern analysis and G418 resistance. Analysis of the steady-state RNAs produced after transfection of the packaging cell lines showed that MFG expressed a significantly higher level of genomic RNA, which contains the packaging signal, than the other vectors while still expressing a high level of the subgenomic RNA encoding CAT. The high level of genomic RNA most likely contributes directly to the higher titer of MFG. We also compared viral titers from subcloned PA317 producer lines containing LNC-CAT and MFG-CAT-Neo, and confirmed that the titer of the MFG virus was higher than that of the LNCX. In selected subcloned transduced NIH3T3 cells, average levels of CAT activity were nine-fold higher from MFG-based vector. Our results suggest that there are significant differences in both the titer and the level of gene expression between retroviral vectors which are currently being used in gene therapy clinical trials.