The development of autonomic neuronal precursors was studied in cultures of microsurgically excised quail neural crest grown alone and associated with other young embryonic tissues. Biochemical differentiation in the cultures was followed by measuring their ability to synthesize acetylcholine (ACh) and catecholamines (CA) from radioactive precursors; cytochemical aspects of their differentiation were examined by techniques including electron microscopy, cholinesterase histochemistry, and CA cytofluorescence. Mesencephalic crest, which can make ACh before explantation, always synthesized ACh after 7 d in culture and often, but not invariably, elaborated small quantities of CA as well. Association with 2-d somite and notochord, 2-d heart, or 4-d hindgut, in medium supplemented with horse serum, resulted in the synthesis of increased amounts of both transmitters. ACh-synthesizing activity was lower and the cholinergic-stimulating effects of somite and heart were abolished in the presence of fetal calf serum. Cervicothoracic (trunk) crest, taken from the level where the dorsal mesoderm is still unsegmented, always produced ACh after culture, but CA was detectable only when the cultures were obtained by initially explanting the entire neural primordium. Co-culture of trunk crest with young embryonic tissue increased ACh-synthesizing ability and initiated CA production. Despite their capacity to elaborate neurotransmitter, cultures of either type of neural crest, alone or in association with the above-mentioned tissues, contained very few cells resembling neurons in their phase contrast appearance and none that reacted positively to any of the cytological tests applied. On the other hand, when the sclerotomic moiety of 3-d somite was cultured, trunk neural crest cells that had already migrated into the rudiment in vivo but which had not yet begun to produce detectable amounts of CA underwent rapid differentiation into neurons that synthesized and accumulated large quantities of CA. Stores of CA were detectable cytochemically as early as 24 hr after explantation and the presence of many small, dense core vesicles in neurons and processes was revealed by electron microscopy. ACh-synthesizing activity, demonstrable in freshly dissected sclerotomes, was also present in all of the cultures examined. These results show that (1) during ontogeny, cholinergic traits appear earlier than adrenergic ones in the neuronal precursors contained in the neural crest; (2) some decisive step in the differentiation of the precursor cells of the sympathetic ganglia takes place in vivo within a few hours of the onset of trunk neural crest migration. This coincides with a maturation of the somitic mesenchyme. A similar developmental process does not occur in vitro when 2-d somites and neural crest are associated in histiotypic cultures.