Tissue-specific transgene expression is a valuable research tool and is of great importance in delivering toxic gene products with adenovirus vectors to tumors. Limiting cytotoxic gene expression to the target cells is highly desirable. While a number of successful applications of tissue- and tumor-specific gene expression using Ad vectors has been reported, cloning of some promoters into Ad vectors resulted in modulation or loss of tissue specificity. This phenomenon is likely the result of the interaction of E1A enhancer (and possibly other Ad sequences) with the promoter cloned in the E1 region. We have compared performance parameters of prostate-specific and tet-regulatable promoters in plasmids containing the terminal repeat sequences of Ad5 with or without the E1A enhancer. Subsequently, adenoviral vectors were constructed containing identical expression units either in the E1 region or near the right ITR, and tested in several cell lines. Here, we report that promoters placed near the right ITR of Ad5 retain higher selectivity and lower background expression in both plasmid and adenovirus vectors. We confirm that the E1A enhancer can interfere with the desired activity of nearby promoters, and describe an alternative transgene insertion site for construction of Ad vectors.