The choice of transmitter made by postmitotic rat sympathetic neurons in cell culture can be controlled by the environment in which they develop. One of the differentiation signals is a protein secreted by heart cells that can induce previously noradrenergic neurons to synthesize acetylcholine and form cholinergic synapses. This change in phenotype occurs without alteration in neuronal survival or growth. The differentiation factor has now been purified at least 100,000-fold, and it is homogeneous by several criteria. (i) The cholinergic activity comigrates with a single 125I-labeled protein band of 45 kDa in one-dimensional NaDodSO4/PAGE. (ii) The biological activity comigrates precisely with a series of five 125I-labeled protein spots of 45 kDa in two-dimensional gel electrophoresis. (iii) Treatment of the 45-kDa band with endo-beta-N-acetylglucosaminidase F reduces the apparent molecular size of both the labeled protein and the biological activity to a band of 22 kDa. The data suggest that the differentiation factor is a slightly basic glycoprotein with at least six glycosylation sites.