Neurotrophic factors (NTFs) can protect against or sensitize neurons to excitotoxicity. We studied the role played by various NTFs in the excitotoxic death of purified embryonic rat motor neurons. Motor neurons cultured in brain-derived neurotrophic factor, but not neurotrophin 3, glial-derived neurotrophic factor, or cardiotrophin 1, were sensitive to excitotoxic insult. BDNF also induces excitotoxic sensitivity (ES) in motor neurons when BDNF is combined with these other NTFs. The effect of BDNF depends on de novo protein and mRNA synthesis. Reagents that either activate or inhibit the 75-kDa NTF receptor p75NTR do not affect BDNF-induced ES. The low EC50 for BDNF-induced survival and ES suggests that TrkB mediates both of these biological activities. BDNF does not alter glutamate-evoked rises of intracellular Ca2+, suggesting BDNF acts downstream. Both wortmannin and LY294002, which specifically block the phosphatidylinositol 3-kinase (PI3K) intracellular signaling pathway in motor neurons, inhibit BDNF-induced ES. We confirm this finding using a herpes simplex virus (HSV) that expresses the dominant negative p85 subunit of PI3K. Infecting motor neurons with this HSV, but not a control HSV, blocks activation of the PI3K pathway and BDNF-induced ES. Through the activation of TrkB and the PI3K signaling pathway, BDNF renders developing motor neurons susceptible to glutamate receptor-mediated cell death.