In certain applications of gene therapy, regulated (rather than constitutive) gene expression will be crucial for proper function or optimal effect of the delivered gene. Therefore, an important goal of gene therapy is to be able to deliver genes so that they express in a pattern that recapitulates the expression of an endogenous cellular gene. Additionally, regulating transgene expression will be an important component of efforts for reducing risks of adverse advents associated with gene therapy with integrating vectors. Although tissue-specific promoters confer selectivity, in a vector- based system, their activity may be too weak to mediate sufficient levels of gene expression to achieve therapeutic efficacy in vivo. Additionally, amplification of a specific, but weak, signal would be beneficial to some applications, such as imaging. Iyer et al. (PNAS, 2001) described a two-step transcriptional activator (TSTA) system to amplify gene expression while preserving tissue specificity using adenoviral vectors. In this system, the tissue-specific promoter drives expression of a potent synthetic transcription activator (the yeast GAL4 DNA binding domain fused to the activation domain of the HSV-1 VP16 activator), which in turn activates a GAL4-responsive reporter. We have developed lentivirus-based vectors using this 2-tiered system with the constitutive but weak PGK housekeeping gene promoter and the human insulin promoter and have tested them in cell lines and primary cells. Compared to a vector carrying only the PGK promoter to drive eGFP expression, the PGK amplifiable- vector amplified eGFP expression from 2–11 fold, depending on the cell type. Vectors carrying the insulin promoter did not express in non-β cell lines (HT29, HEK293), but expressed in Min6 cells, a murine insulinoma cell line, indicating that the insulin promoter was capable of conferring cell specificity. Additionally, the insulin amplifiable-vector was able to amplify expression 4.6 times in these cells. In primary murine islets, no gene expression was detected from the vector carrying only the insulin promoter, but low eGFP expression was detected from the insulin-promoter amplifiable vector. Thus, we have demonstrated that it is possible to build lentivirus vectors carrying an amplifiable gene expression system that retains tissue specificity. These vectors will be useful in gene expression studies that require a detectable signal and tissue specificity.