Tumor-specific expression of therapeutic genes is a prerequisite in many approaches to retrovirus-mediated cancer gene therapy. However, tissue specificity is often associated with a reduction in viral titer. To overcome this problem, we constructed a series of murine leukemia virus (MLV)-based retroviral promoter conversion (ProCon) vectors carrying either the simian virus 40 poly(A) signal trimer (3pA) inserted in the 3' long terminal repeat (LTR) of these vectors or the human cytomegalovirus enhancer region (CMVe) inserted 5' and 3' of the retroviral LTRs. Furthermore, an extended AT stretch/attachment site (AT/att) of wild-type MLV was introduced into the vector. In the vector-producing cells, insertion of the CMVe and/or the 3pA resulted in a three- to fourfold-enhanced marker gene expression compared to the parental vector, whereas insertion of the AT/att gave a slight decrease in expression. The combination of all three modifications had no additional effects. In contrast, however, neomycin selection of infected cells revealed only a slight increase in virus titer with vectors carrying the 3pA modification; the titer was increased by 1 with vectors containing the extended AT/att, although the viral DNA copy numbers in infected cells were similar with both types of vectors. Thus, insufficient integration rather than insufficient reverse transcription and/or production of virus RNA is the major cause for the low titer obtained with the ProCon vectors. The combination of all three modifications resulted in a 2- to 3-log increase in the virus titer. These modifications result in expression targeted ProCon vectors with titers similar to those of nonmodified MLV-based vectors.