Two key components of neural development and regeneration, survival of the involved neurons and elongation of neuritic elements, are likely to depend on the availability of an appropriate trophic drive to these neurons. At present, only one trophic factor, Nerve Growth Factor, is known to ensure both survival and neuritic growth for its target neurons. A search for a second such agent, a putative cholinergic neuronotrophic factor (CNTF), has been undertaken using as indicators neuronal survival, neurite outgrowth and choline acetyltransferase (CAT) activity in monolayer cell cultures. Eight-day chick embryo ciliary ganglia yielded two monolayer culture systems which appear to be well suited for a CNTF assay. Ciliary ganglionic dissociates, seeded on a highly adhesive collagen substratum, show no neuronal survival by 24 h if the medium is supplemented only with serum or chick embryo extract. However serum and embryo extract combined support survival of, and extensive neuritic outgrowth from, nearly the theoretical number of ganglionic neurons seeded. Alternatively, ciliary ganglionic neurons can be made to survive and produce a profuse neuritic outgrowth on polyornithine-coated dishes if supplied with medium conditioned over chick embryo heart muscle cultures, as already described by other laboratories. The two trophic sources differ markedly in their effects on the ganglionic neurons when tested on collagen or polyornithine substrata, and in some cases when different serum supplements are used. Neuronal survival, neurite production and, possibly, CAT activity appear to be subject to independent regulation. The culture systems used in this study can be developed into quantitative bioassays for the isolation of the different agents responsible for neuronal survival and neurite promotion, and for the investigation of their activities.