Retroviral vectors have been widely used in gene therapy due to their simple genomic structure and high transduction efficiency. We report a construction of Moloney murine sarcoma virus (MoMSV) and Moloney murine leukemia virus (MoMLV) hybrid-based retroviral vectors with significantly improved efficiency of transgene expression after stable incorporation into the host genome. In these vectors, the residual gag gene coding sequence located in the extended region of packaging signal was removed. These vectors, therefore, contain no coding sequence for the gag, pol, or env gene that can be used for homologous recombination with sequences introduced in the packaging system for a recombinant competent retrovirus (RCR) generation. A strong splice acceptor site obtained from the exon/intron junction of either the chimpanzee EF1-alpha gene or the human CMV major immediate early gene was placed downstream of the MoMSV packaging signal (Psi), significantly improving the efficiency of transgene expression. The 5' LTR U3 sequence was replaced with an extended human CMV major immediate early gene enhancer/promoter for a strong expression of full-length messages from the viral backbone, helping to maintain high levels of viral titer. These newly developed retroviral vectors should facilitate RCR-free gene transfer with significantly improved efficacy in clinical gene therapy trials.