Serotonin (5HT), one of the principal neuromodulators in the mammalian brain, is implicated in a variety of disorders such as pain, depression and schizophrenia. Many aspects of serotonergic transmission remain unknown, necessitating the development of new research tools. Previously, we successfully used viral vectors for cell-specific gene expression in the brain in order to selectively study or modulate the function of targeted neurones (Duale et al. 2007; Wang et al. 2006; Chiti & Teschemacher, 2007). Here we present novel lenti- and adenoviral vectors suitable for selective gene expression in raphe 5HT neurones. For targeting we used partial sequences (length 3.6kb, 2kb, and 1kb) of the natural promoter of rat tryptophan hydroxylase 2 (TPH2), the rate limiting enzyme in 5HT synthesis, obtained by PCR from rat brain genomic DNA. Lentiviral vectors for expression of EGFP were prepared using standard protocols (Liu et al. 2008) and stereotaxically microinjected into the rat raphe nuclei (under a mixture of ketamine (60 mg/kg) and medetomidine (250 µg/kg) i.m. anaesthesia). Specificity was then determined by immunofluorescence using anti-GFP and anti-TPH2 antibodies. The 3.6kb and 2kb promoter sequences conferred specific expression (co-localisation >95%), while the specificity of the 1kb promoter was only ~78%. However, native promoters were weak, and expression could only be detected using anti-GFP antibodies. To overcome this limitation, we employed a previously established transcriptional amplification strategy which involves cell-specific co-expression of a potent chimeric transactivator (Liu et al. 2008; Liu et al. 2006). This strategy increased the potency of 3.6kb and 2kb TPH2 promoters, leading to visible EGFP expression, while maintaining 5HT neurone specificity at 99% (n= 700 cells). Adenoviral vectors based on the 3.6kb construct were generated which caused visible EGFP expression in 5HT neurones in organotypic brainstem slice cultures. Moreover, it was possible to visually identify EGFP-positive axons with multiple small varicosities. Using previously established methods (Chiti & Teschemacher, 2007), we made the first microamperometric recordings of quantal 5HT release, and the first patch clamp recordings from EGFP-expressing 5HT neurones of the rat raphe. We believe that these viral vectors have great potential for in vivo and in vitro studies into the function of central 5HT neurones.