Environmental cues play an important role in determining the transmitter phenotype of developing sympathetic neurons. Several factors have been described which can induce cholinergic function in cultured sympathetic neurons. We have compared certain biological and immunological properties of three of them, cholinergic differentiation factor (CDF), membrane-associated neurotransmitter-stimulating factor (MANS), and ciliary neurotrophic factor (CNTF), to determine whether they are different. As previously reported, all three increased acetylcholine synthesis in cultured sympathetic neurons. In addition, MANS as well as CNTF and CDF decreased catecholamine synthesis. CNTF and MANS, but not CDF, promoted the survival of embryonic chick ciliary neurons. Affinity-purified antibodies raised against a synthetic peptide corresponding to the N-terminal sequence of CDF immunoprecipitated CDF, but not MANS or CNTF. These results indicate that although CDF, MANS, and CNTF have similar effects on transmitter synthesis by cultured sympathetic neurons, CDF lacks the ciliary neurotrophic activity of MANS and CNTF. Further, CDF possesses an N-terminal epitope which is absent from both MANS and CNTF. Thus, CDF is distinct from MANS and CNTF, and at least two factors exist which can alter the transmitter phenotype of sympathetic neurons in vitro.