A persistent obstacle in the use of vector systems for gene therapy has been the inability to attain high-level expression of the target gene in primary cells in vivo. The MFG retroviral vector was designed to yield improved expression over the widely used N2 or LN vectors; however, the molecular basis for this effect has not been examined. Using the human glucocerebrosidase (GC) enzyme as a reporter, we compared expression from the MFG and N2 vector backbones in transduced murine hematopoietic cells after syngeneic bone marrow transplantation. Reporter enzyme activities in primary spleen colonies of transplanted mice were seven-fold higher per vector copy in cells transduced with the (MFG-based) MGC vector than in cells bearing the (N2-based) G2 vector. In spleen colonies harboring the MGC vector, the ratio of spliced to unspliced vector RNA was increased four-fold relative to the G2 vector transcripts in Northern blot analyses. Further analyses indicated that MGC-transduced cells contained five-fold higher levels of spliced RNA per vector copy. Since translation of spliced RNA species (in which the complex secondary structure of the packaging signal has been excised) is likely to proceed with enhanced efficiency, the augmented levels of spliced RNA produced by MFG may represent the key element of increased protein expression from this vector. These findings suggest that the MFG retroviral vector may provide higher level expression of target genes used in human gene therapy.