Cardiac function is modulated by norepinephrine release from innervating sympathetic neurons. These neurons also form excitatory connections onto cardiac myocytes in culture. Here we report that brain-derived neurotrophic factor (BDNF) altered the neurotransmitter release properties of these sympathetic neuron-myocyte connections in rodent cell culture, leading to a rapid shift from excitatory to inhibitory cholinergic transmission in response to neuronal stimulation. Fifteen minutes of BDNF perfusion was sufficient to cause this shift to inhibitory transmission, indicating that BDNF promotes preferential release of acetylcholine in response to neuronal stimulation. We found that p75(-/-) neurons did not release acetylcholine in response to BDNF and that neurons overexpressing p75 showed increased cholinergic transmission, indicating that the actions of BDNF are mediated through the p75 neurotrophin receptor. Our findings indicate that p75 is involved in modulating the release of distinct neurotransmitter pools, resulting in a functional switch between excitatory and inhibitory neurotransmission in individual neurons.