Although brain-derived neurotrophic factor is the most abundant and widely distributed neurotrophin in the nervous system, reproducible determinations of its levels have been hampered by difficulties in raising suitable monoclonal antibodies. Following immunization of mice with recombinant fish and mammalian brain-derived neurotrophic factor, monoclonal antibodies were generated and used in an immunoassay based on the recognition of two different epitopes. Neither antibody crossreacts with neurotrophin homodimers other than brain-derived neurotrophic factor, although reactivity was detected with brain-derived neurotrophic factor/neurotrophin-3 heterodimers. As both nerve growth factor and neurotrophin-3 are known to affect the development of a variety of neurons expressing the brain-derived neurotrophic factor (bdnf) gene, this assay was used to determine levels in tissues isolated from newborn mice carrying a null mutation in the nerve growth factor (ngf) or the neurotrophin-3 (nt3) gene. Marked differences were observed between mutants and wild-type littermates in the PNS, but not in the CNS, suggesting that neither nerve growth factor nor neurotrophin-3 is a unique regulator of brain-derived neurotrophic factor levels in the newborn mouse CNS.