To explore the role of neurotrophin-3 (NT-3) during cerebral ischemia, NT-3-deficient brains were subjected to transient focal ischemia. Conditional mutant brains produced undetectable amounts of NT-3 mRNA, whereas the expression of the neurotrophin, BDNF, the NT-3 receptor, TrkC, and the nonselective, low-affinity neurotrophin receptor p75NTR, were comparable to wild-type. Baseline absolute blood flow, vascular and neuroanatomical features, as well as physiological measurements were also indistinguishable from wild-type. Interestingly, the absence of NT-3 led to a significantly decreased infarct volume 23 h after middle cerebral artery occlusion. Consistent with this, the addition of NT-3 to primary cortical cell cultures exacerbated neuronal death caused by oxygen-glucose deprivation. Coincubation with the oxygen free radical chelator, trolox, diminished potentiation of neuronal death. NT-3 also enhanced neuronal cell death and the production of reactive oxygen species caused by oxidative damage inducing agents. We conclude that endogenous NT-3 enhanced neuronal injury during acute stroke, possible by increasing oxygen-radical mediated cell death.