There is now widespread evidence indicating that nerve growth factor (NGF) is involved in the function of central cholinergic neurons. Its possible role in the establishment of cholinergic fiber connectivities was studied in co-cultures of rat septum and hippocampus. Application of 100 ng/ml NGF greatly increased the number of acetylcholinesterase (AChE)-positive fibers which invaded the hippocampal slices, an effect which was accompanied by a more than 6-fold elevation of the two major cholinergic enzymes, choline acetyltransferase and AChE. In contrast, application of anti-NGF antiserum, but not a non-immune serum, reduced the number of AChE-positive fibers which grew into and remained within the hippocampal slices. Since no diffuse outgrowth of AChE-positive fibers from the septum was observed following application of NGF, these results suggest that NGF plays a role in the stabilization and long-term maintenance of the cholinergic septo-hippocampal projection in vitro.