A purified polyclonal antibody preparation was made against recombinant brain-derived neurotrophic factor (BDNF) in guinea pig and characterized for use in immunoassays and immunohistochemistry. The anti-BDNF antibodies specifically recognized BDNF in Western blots and immunoprecipitation. There was no cross-reactivity with the other known mammalian members of the neurotrophin family, nerve growth factor, neurotrophin-3 and neurotrophin-4/5. In immunohistochemical analysis, the anti-BDNF recognized exogenous BDNF injected into the brain of rats, whereas no signal was obtained with the other neurotrophins. Preabsorption with native BDNF abolished the immunoreactivity in brain sections. These studies identify the anti-BDNF as a tool for immunocytochemistry and the development of an immunoassay. Immunohistochemical analysis revealed widespread neuronal localization of BDNF in many brain areas. BDNF was localized in all subpopulations of hippocampal neurons. The distribution in the hippocampus suggests localization in the cytoplasm of cell bodies and dendrites.