The neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) are known to exist in solution as non-covalently linked homodimeric proteins. The recent elucidation of the crystal structure of the NGF homodimer, as well as the conservation of structural motifs in the neurotrophins, raised the possibility that neurotrophin heterodimers might also occur. The formation of BDNFNT-3 heterodimers was explored using a vaccinia virus expression system. Upon co-infection of cells with viruses expressing BDNF and NT-3, we could identify a BDNFNT-3 heterodimer as a biosynthetic product and separate it from the BDNF and NT-3 homodimers. We could also show that the BDNF/NT-3 heterodimers can be formed irrespective of wild-type or exchanged prosequences, indicating that prosequence specificity does not influence dimer formation. In all neuronal survival assays that were used, the BDNFNT-3 heterodimer was shown to be 10-fold less active compared with a mixture of BDNF and NT-3 homodimers. This lower specific activity was also measured in a neuronal population co-expressing receptors for BDNF and NT-3. The low biological activity of the heterodimer observed with neurons was not paralleled by a reduced ability of the heterodimer to interact with trkB or trkC receptors, as assessed by the induction of tyrosine phosphorylation of these receptors expressed by fibroblast cell lines.