Viral vectors have become important tools to effectively transfer genes into terminally differentiated cells, including neurons. However, the rational for selection of the promoter for use in viral vectors remains poorly understood. Comparison of promoters has been complicated by the use of different viral backgrounds, transgenes, and target tissues. Adenoviral vectors were constructed in the same vector background to directly compare three viral promoters, the human cytomegalovirus (CMV) immediate-early promoter, the Rous sarcoma virus (RSV) long terminal repeat, and the adenoviral E1A promoter, driving expression of the Escherichia coli lacZ gene or the gene for the enhanced green fluorescent protein. The temporal patterns, levels of expression, and cytotoxicity from the vectors were analyzed. In sensory neuronal cultures, the CMV promoter produced the highest levels of expression, the RSV promoter produced lower levels, and the E1A promoter produced limited expression. There was no evidence of cytotoxicity produced by the viral vectors. In vivo analyses following stereotaxic injection of the vector into the rat hippocampus demonstrated differences in the cell-type-specific expression from the CMV promoter versus the RSV promoter. In acutely prepared hippocampal brain slices, marked differences in the cell type specificity of expression from the promoters were confirmed. The CMV promoter produced expression in hilar regions and pyramidal neurons, with minimal expression in the dentate gyrus. The RSV promoter produced expression in dentate gyrus neurons. These results demonstrate that the selection of the promoter is critical for the success of the viral vector to express a transgene in specific cell types.The ability to introduce foreign genes into nondividing cells, such as neurons, has benefited from the availability of viral vectors that include recombinant herpes simplex virus vectors (9,17,23,26), herpes simplex virus-based amplicons (18, 19), adenovirus vectors (6, 7, 12, 29), vaccinia virus-based vectors (11, 24), adeno-associated virus vectors (2,16,21,27), and lentivirus vectors (5,37,38). Virtually all of these vectors have provided the opportunity to modify gene function, although the efficiency of transduction, the cytotoxicity, and the patterns of gene expression vary considerably.Adenoviral vectors have been used extensively for gene transfer into neurons. The human cytomegalovirus (CMV) immediate-early promoter, the Rous sarcoma virus (RSV) long terminal repeat, and the adenovirus E1A promoter have been successfully used in adenovirus vectors to drive expression of foreign genes in neurons (6,7,12,28,29,45). Direct comparison of the efficiency of the adenovirus-mediated gene transfer has been complicated by differences in the adenovirus vector background, the wide range of targeted cell types, and complications caused by immune responses in animal studies. Therefore, the extent to which the promoter determines the efficiency of transgene expression in the context of the viral vector remains lar...