Cultures were prepared by dissociating 3-day-old whole chick embryos, or 6- or 8-day-old chick embryo cerebral hemispheres. The dispersed cells were plated onto poly-L-lysine coated dishes in Dulbecco's modified Eagle's medium fortified with 10% fetal bovine calf serum. Immunocytochemistry, histochemistry and biochemistry were used to characterize the cholinergic neurons in these cultures. Double staining for acetylcholinesterase (AChE) and neurofilament revealed that a high percentage of neurons also contained AChE; fewer neurons though were positive for choline acetyltransferase (ChAT). The presence of AChE in non-cholinergic neurons was interpreted to be indicative of 'cholinoceptive' neurons in culture. The developmental profile for ChAT activity in cultures derived from 3-day-old whole chick embryos was previously described; we have reported that activity reached high levels by 7 days in culture. In this study we found that in cultures derived from 6-day-old chick embryos ChAT activity increased slowly up to 6 days in culture and rose markedly thereafter; in contrast, in cultures derived from 8-day-old chick embryos activity was low until day 9 and rose moderately thereafter. The differences in ChAT expression between cultures derived from chick embryos of different developmental stages may reflect a loss of plasticity of the cholinergic population with age.