Expression of multiple genes from the same target cell is required in several technological and therapieutic applications such as in vivo tracking of stem cells. In spite of such need, reaching independent, coordinate and high level dual-gene expression cannot be reliably accomplished by current gene transfer vehicles. To address this question, we designed and optimized a lentiviral vector carrying two internal promoters each driving expression of a single gene independent of the other. The two promoters were spaced by a synthetic polyA, as well as terminator and insulator sequences. A second insulator sequence was cloned downstream of the polyA of the second gene to alleviate the potential suppression of gene expression from the viral enhancer. All the internal transcription units were cloned in trans to avoid interference of viral mRNA production by the inserted polyAs. With this design, we found that more than 90% transduced cells by this vector co-expressed both genes and that the expression level of both genes was as high as that yielded from lentiviral vectros containing only a single transcriptional unit. Furthermore, the performance of the newly designed vector was independent of cell types and promoters used. Using this vector, we demonstrated tissue specific gene transfer from promoters specific for smooth muscle cells or epidermal keratinocytes. We also demonstrated that this vector can be used for dynamic, real-time monitoring of gene expression suggesting that it may be very useful in high throughput gene expression profiling studies.