The effects of nerve growth factor (NGF) on the intracellular content of acetylcholine (ACh) in cultured septal neurons from developing rats have been examined. The content of ACh could be measured by using HPLC and electrochemical detection (HPLC-ECD), coupled with an immobilized enzyme column. This method of determination is very simple and rapid, and is highly sensitive. The content of ACh and the activity of choline acetyltransferase (ChAT) in cultured postnatal day 1 (P1) septal neurons grown on an astroglial "feeder" layer was increased during the period of cultivation by the addition of NGF. The activities of ChAT and the content of ACh increased in a dose-dependent manner in direct relationship to the different amounts of NGF employed. These effects of NGF, i.e., elevating the intracellular content of ACh, accompanied by an increase in activity of ChAT, also were confirmed in the P1 septal organotypic cultures. Additionally, embryonic day 17 (E17) septal neurons in a serum-free medium displayed a similar responsiveness to NGF with respect to the elevation in the content of ACh and the increase in activity of ChAT. These results suggest that intracellular levels of ACh are