Targeting therapeutic transgene expression to defined tissues is a major task in the development of safe and efficient gene therapy protocols. Recombinant adenovirus is an attractive vector because it can be prepared in huge quantity and new generation vectors possess very large cloning capacities combined with reduced immunogenicity. In the brain, adenovirus transduces mainly glial cells, making it difficult to use this vector system in applications that need expression of therapeutic proteins in neurons. Here, we show that by using a small fragment of the human synapsin 1 gene promoter, we were able to restrict transgene expression from an adenoviral vector exclusively to neurons. Furthermore, we obtained stable long-term transgene expression from this vector in striatum and thalamus at appropriate vector dose. Other promoters like the CMV and U1snRNA promoters also mediated transgene expression over several months, but mainly in glial cells. Although the NSE promoter was relatively neuron specific, it still expressed in glial cells also, and was clearly outperformed by the synapsin promoter with respect to transcriptional neuronal targeting. As an important feature of adenoviral-mediated gene transfer to the brain, we demonstrate that dopaminergic neurons of the substantia nigra do not allow for long-term expression from adenoviral vectors. Strikingly, these neurons appeared to specifically attenuate transgene expression by deleting the adenoviral genome.