Neuronal depolarization and culture media conditioned by certain nonneuronal cells (CM) are known to exert opposite effects on the expression of cholinergic and noradrenergic traits in cultured rat sympathetic neurons. We have compared their effects on the developments of choline acetyltransferase (CAT), tyrosine hydroxylase (TOH), dopa decarboxylase (AADC) and acetylcholinesterase (AcChE) in these cultures. A macromolecular factor which was partially purified from CM increased CAT development in a dose-dependent manner and depressed the development of TOH and AADC by 5- to 10-fold. In the presence of intermediate concentrations of this partially purified factor, both CAT and catecholamine synthesizing enzymes developed to high levels, whereas high concentrations caused a long-lasting, but not total, impairment of TOH development. The effects of CM on both CAT and AADC activities resulted from variations in the number of immunotitratable enzyme molecules. Conversely, K+ ions (30-40 mM) depressed the development of CAT by 90% and stimulated TOH development 2.5-fold. Cultures grown with CM in high K+ medium had similar CAT and TOH activities as compared to those cultures grown without CM in low K+ medium suggesting that CM and K+ ions had antagonistic effects on the expression of these enzymes. However, K+ ions did not affect the development of AADC in these cultures. CM suppressed in a reversible manner the development of the 16 S form of AcChE. In the presence of 40 mM K+, the rate of development of AcChE was reduced. In particular, the development of 16 S AcChE was strikingly impaired, although not totally suppressed. The effect of elevated K+ ions on the percentage of 16 S AcChE was rapidly reversible. It is concluded that CM and elevated K+ ions have antagonistic effects on CAT and TOH, but not on AADC development; AcChE, in particular its asymmetric 16 S form, is regulated independently of the cholinergic/noradrenergic status of sympathetic neurons.