Cultures were prepared by dissociating 3-day-old whole chick embryos and plating the dispersed cells on poly-L-lysine-coated dishes in Dulbecco's Modified Eagle's Medium with 10% fetal calf serum. By 48 hr in culture, aggregates and neuritic sprouting were observed. Long neuritic bundles connecting cell aggregates were evident by 4 days in culture. Consistent patterns throughout the lifespan of the cultures were contacts between neurites, and flat isolated cells, presumptively glial, emerged. Throughout the lifespan of the cultures, the cholinergic cell population was characterized histochemically by the method of Karnovsky and Roots and biochemically by assaying choline acetyltransferase. By 4 days in culture, all aggregates showed light cholinesterase-positive staining; however, with days in culture, several aggregates had no staining, and some positive-stained aggregates were interconnected with other aggregates showing only spotted positive staining. Choline acetyltransferase activity showed a developmental profile in agreement with the histological findings. The early presence of choline acetyltransferase activity is taken as indication of the early commitment of cholinergic neurons.