We have found that cholinergic neurons in spinal cord-dorsal root ganglion cultures derived from E12-E13 mouse embryos are sensitive, as measured by changes in choline acetyltransferase activity, to factors secreted by non-neuronal cells derived from the same tissue at an identical developmental stage. Conditioned medium was produced by incubating non-neuronal cultures for 4 days in defined medium. The cholinotrophic activity present in the conditioned medium had a molecular weight of greater than 50,000 as determined by ultrafiltration and bound wheat germ lectin and heparin sepharose. Total RNA isolated from the non-neuronal cells, used to produce the conditioned medium, was translated in frog oocytes. Conditioned medium from the injected oocytes was also found to contain cholinotrophic activity. In contrast, the conditioned medium from water-injected oocytes was inactive. The interaction between the cholinotrophic activity in conditioned medium from frog oocytes and known second messengers was also examined. Dibutyryl cyclic AMP produced a concentration-dependent increase in choline acetyltransferase activity. If a maximal effective dose of dibutyryl cyclic AMP was added in conjunction with a maximal effective dose of conditioned medium from oocytes injected with total RNA a nearly additive response was noted. In contrast, the phorbol ester, phorbol 12-myristate 13-acetate, produced a biphasic change in the level of choline acetyltransferase activity; with lower doses stimulating and higher doses inhibiting the enzyme activity. When conditioned medium from oocytes injected with non-neuronal cell RNA was added in conjunction with the phorbol ester a decrease in the physiological response was noted.