A diphtheria toxin‐neurotrophin‐4/5 (NT‐4/5) chimera (DAB389‐NT4), in which the native receptor binding domain of diphtheria toxin was replaced with a synthetic gene encoding rat NT‐4/5, was expressed, refolded, and purified. This fusion toxin has a deduced molecular mass of 60,163 and is formed by joining the first 389 amino acids of diphtheria toxin to amino acids 1–130 of mature rat NT‐4/5, using an NH2‐terminal bridge of 33 additional amino acids including six consecutive histidines. Neural cell types expressing only p75LNGFR or p75LNGFR and full‐length or truncated TrkB were used to evaluate the cytotoxic efficacy of DAB389‐NT4. The fusion toxin produced a concentration‐dependent killing of all cell populations, with LC50 values that largely reflected the known NT‐4/5 binding affinities for these receptor proteins. Mean LC50 values ranged from 2,960 pM in p75LNGFR‐expressing neuro‐2a neuroblastoma cells to 1,075 and 70 pM, respectively, in hippocampal astrocytes (p75LNGFR+/truncated TrkB+) and cerebellar granule cells (p75LNGFR+/TrkB+). The LC50 for DAB389‐NT4 in receptor‐negative 3T3 fibroblasts was 20 nM. NT‐4/5 and brain‐derived neurotrophic factor but not ciliary neurotrophic factor added in excess neutralized DAB389‐NT4 cytotoxicity. NT‐4/5, however, did not reduce the cytotoxicity of intact diphtheria toxin.