Although developing motor neurons express low-affinity nerve growth factor (NGF) receptors, there is no known biological effect of NGF on developing or adult motor neurons. In this study, we found that, unlike NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT-4/5) stimulated cholinergic phenotype by increasing choline acetyltransferase (CAT) activity in cultures enriched with embryonic rat motor neurons. Ciliary neurotrophic factor (CNTF) also stimulated CAT activity. The effects of BDNF and NT-4/5 on CAT activity appeared to be synergistic with that of CNTF. Cotreatment with BDNF and NT-3 resulted in an additive effect, suggesting that signal transduction was mediated through different high-affinity receptors tyrosine kinases B and C (Trk B and Trk C). However, cotreatment with BDNF and NT-4/5 did not result in an increase in CAT activity greater than that of either BDNF or NT-4/5 alone, suggesting that their effects were mediated via the same receptor Trk B. Supporting our findings that spinal cholinergic neurons are responsive to trophic actions of members of the neurotrophin family, motor neuron-enriched cultures were found to express mRNA for Trk B and Trk C, which have been identified as high-affinity receptors for BDNF and NT-4/5, and NT-3, respectively.