Delivery of viral vectors encoding the Cre recombinase is showing promise to target gene modification in specific brain regions. Here we describe the targeting of the dorsal raphe nucleus (DRN), which contains the majority of the serotonin (5-HT) neurons projecting to the forebrain. First, we demonstrate successful transgene expression in the mouse DRN by stereotaxic delivery of the AdnlslacZ adenoviral vector. Second, we show that expression of the Cre recombinase can be achieved in the 5-HT neurons by optimized injection of the Adcre vector. Using reporter mice in which Cre activity induces beta-galactosidase (beta-gal) expression, we demonstrate efficient Cre-mediated recombination and persistence of beta-gal positive 5-HT neurons at least 1 month postinjection. Together, these results demonstrate that viral delivery provides a valuable method to target Cre recombination throughout the murine DRN and thus to study 5-HT neurotransmission by conditional gene modification.