We examined the development of cholinergic neuronal functions and AMPA response in cultured embryonic rat septal cells. Choline acetyltransferase activity was detectable from 4 to 6 days in culture and reached to a plateau at day 8. Acetylcholine (ACh) release was detected at day 8 and the release was inhibited by treatments of omega-conotoxin, tetrodotoxin, vesamicol (a vesicular ACh transporter inhibitor) and CNQX (an AMPA receptor antagonist). Therefore, the ACh release is due to neuronal activities and AMPA receptor-mediated excitatory input is involved in ACh release. AMPA-induced increase in intracellular Ca 2 + level was observed at day 3 and most of AMPA-responsive cells coincided with high-K + responsive cells. These results suggest that cholinergic neurons develop their neuronal functions about day 8 under the cultural conditions, and expression of AMPA receptors precedes the cholinergic functional development.