The high vocal center (HVC) controls song production in songbirds and sends a projection to the robust nucleus of the archistriatum (RA) of the descending vocal pathway. HVC receives new neurons in adulthood. Most of the new neurons project to RA and replace other neurons of the same kind. We show here that singing enhances mRNA and protein expression of brain-derived neurotrophic factor (BDNF) in the HVC of adult male canaries, Serinus canaria. The increased BDNF expression is proportional to the number of songs produced per unit time. Singing-induced BDNF expression in HVC occurs mainly in the RA-projecting neurons. Neuronal survival was compared among birds that did or did not sing during days 31-38 after BrdUrd injection. Survival of new HVC neurons is greater in the singing birds than in the nonsinging birds. A positive causal link between pathway use, neurotrophin expression, and new neuron survival may be common among systems that recruit new neurons in adulthood.A set of interconnected brain nuclei referred to as the song system is responsible for song acquisition and production in songbirds. This system includes the high vocal center (HVC), which sends projections to two nuclei, the robust nucleus of the archistriatum (RA), and Area X (Fig. 1). The main motor pathway for song production runs from HVC to RA; RA in turn innervates mesencephalic and medullary nuclei that control respiratory muscles and the bird's vocal organ, the syrinx (1, 2). The projection from HVC to Area X is part of a separate forebrain circuit that is necessary for song learning but not for the production of learned song (3-6).HVC continues to receive new neurons in adulthood, and a majority of these cells project to RA (7-10). The recruitment of new HVC neurons is part of a replacement process, with peaks in cell death in August (midsummer) and January (midwinter), when blood testosterone levels of adult male canaries are low. These peaks in cell death are followed by peaks in new neuron incorporation in October and March, when blood testosterone levels are high (11). Male canaries modify their song in adulthood by adding, dropping, and altering song syllables. Most of these changes occur during the summer and fall, with a secondary wave of change in winter. Song stability is maximal in the spring, when canaries breed and recruitment of new HVC neurons is at its lowest (12). It has been hypothesized that neuronal replacement in HVC provides a cellular basis for the song plasticity observed in adult canaries (11,13).The mechanism that regulates neuronal survival and turnover in HVC has been the focus of two previous studies. These studies suggested that the survival of new HVC neurons can be regulated by testosterone or a testosterone metabolite (14), and the testosterone effect is mediated by the brain-derived neurotrophic factor (BDNF) (15). Specifically, the increase in the recruitment of new HVC neurons after systemic testosterone treatment is blocked by infusing an antibody against BDNF into HVC. Moreover, the effect of testost...