The effects of Na butyrate on the differentiation of newborn rat sympathetic neurons in primary cultures have been studied. Butyrate did not affect the long-term survival of these neurons in the presence of optimal concentrations of nerve growth factor, but decreased in a dose-dependent manner their protein content. In the range, 0.5-20 mM, butyrate did not modify the specific activity of lactate dehydrogenase in these cultures. Choline acetyltransferase activity developed at a 4.5- to 12-fold higher rate in cultures grown with 1-5 mM butyrate than in its absence. Concomitantly, tyrosine hydroxylase, dopa decarboxylase, dopamine-beta-hydroxylase, and acetylcholinesterase were depressed in cultures grown with butyrate. The deficit in acetylcholinesterase total activity was accompanied by an inhibition of the development of the asymmetric 16 S form of the enzyme. The deficit in tyrosine hydroxylase activity did not result from either a modification of the app Km for the enzyme's cofactor or a modification of its state of cAMP-dependent phosphorylation, but from a decrease in the number of immunoprecipitable enzyme molecules. A similar result was obtained with acetylcholinesterase. Butyrate thus reproduced in a qualitative manner the effects of a macromolecular factor purified from muscle conditioned medium on these neurons (J. P. Swerts, A. LeVan Thaï, A. Vigny, and M. J. Weber (1983) Dev. Biol. 100, 1-11; J. P. Swerts, Le Van Thai, and M. J. Weber (1984) 103, 230-234), raising the hypothesis of a common pathway in the regulation of neurotransmitter phenotype by these two agents.