Transcriptional targeting by specific promoters plays an important role in the development of selective vectors for many gene transfer applications. Many specific promoters have been described (Kasparov et al. 2004), but their applicability is often hindered by their relatively low levels of transcriptional activity compared with stronger but non-selective viral promoters. In this study, we used a recombinant transcriptional amplification approach as a method to enhance transcriptional activity of the weak human SYN1 promoter, which has been shown to drive neuron-specific expression. To this end, SYN1 promoter was used to drive the expression of an artificial transcriptional activator consisting of a DNA binding domain of a yeast transcriptional activator and a strong transactivation domain of a mouse transcriptional factor NF-kB p65. The resultant recombinant transcriptional activator GAL4BD-NF-kB selectively binds to synthetic GAL4 DNA binding sites (GAL4BS) inserted upstream of the second SYN promoter and potently stimulates transcription. Since GAL4BS only exist in yeast genomes and not in eukaryotic genes, there will be no interference for any other gene expressions in transduced mammalian cells. We demonstrate that this method can greatly amplify the expression levels of green fluorescent protein (GFP) and red fluorescent protein (DsRed2) both in vitro and in vivo. In vitro analysis was carried out by transfecting PC12 cells. The transcriptional activity of SYN1 promoter was increased about 10-fold by co-trasnfection with GAL4BD-NF-kB, when the molar ratio of GAL4BD-NF-kB gene expression cassette and that of GFP or DsRed2 was set to 1:1. For in vivo tests, VSV-G pseudotyped lentiviruses were produced using previously published protocol (Coleman et al. 2003). Anaesthetised intramuscularly with a mixture of ketamine (60mg/kg) and medetomidine (250 μg/kg), male Wistar rats were placed in a stereotaxic apparatus. Bilateral injections were made into hypoglossal motor nucleus at three rostro-caudal sites, giving a total of six 1 μl injections. Seven days postinjection, rats were intraperitoneally anaesthetised (sodium pentobarbital, 100mg/kg), perfused and brainstem sections were prepared. Four sections surrounding the injection tract were selected randomly and three fields from each section were used for cell counting. GAL4BD-NF-kB containing viral constructs resulted in ~5-fold higher density of fluorescent cells (expressed by the absolute number of fluorescent positive cells) as compared to conventional SYN1-containing vectors (n = 3). In conclusion, both in vitro and in vivo data indicate that the recombinant transcriptional amplification is a powerful strategy to enhance the activity of the human SYN1 promoter. This strategy can be applied broadly to strongly amplify gene expression of cell or tissue specific promoters, with potential applications for both gene therapy and functional genomic studies.