Neurons dissociated from the embryonic chick superior cervical ganglion (SCG) were separated from ganglionic nonneuronal cells using a density gradient formed with Percoll. The sympathetic neurons were then grown for 3-4 weeks in serum containing medium on a polyornithine substrate precoated with heart-conditioned medium. Both catecholamine (CA) and acetylcholine (ACh) are synthesized and accumulated by these neurons, but the amount of CA is higher and increases much more over time in culture than the amount of ACh. The cultures become therefore more adrenergic with time. We report here that the adrenergic properties of these cells can be enhanced. A 3-fold increase in CA synthesis, as expressed on a per neuron basis, is obtained by increasing neuron cell density 3- to 4-fold. ACh synthesis, however, is decreased at high neuronal density. Optimal CA production is obtained at densities of 120-150,000 neurons/cm2. This effect is due to direct cell contact since it cannot be transferred to low density cultures by medium conditioned by high density cultures. Nerve growth factor concentrations 5-10-fold higher than the amount necessary for optimal neuronal survival (1 microgram/ml 7S NGF) increases CA production but do not affect ACh synthesis. This effect is highest at low plating densities (20-30,000 neurons/cm2, 10-fold increase) and progressively decreases with increasing neuronal density. No increase is obtained in high density cultures where CA production is maximal. In addition, we made the novel observation that medium conditioned by chick liver cells in culture (LCM) increases CA production approximately 4-fold, whereas it does not increase ACh production by the SCG neurons. Work is in progress to biochemically characterize the active component(s) present in the LCM and to determine whether they favor the survival of a subpopulation of adrenergic neurons possible present in these ganglia. Alternatively, the adrenergic differentiation of neurons initially capable of synthesizing both CA and ACh could be selectively increased by LCM.