We have investigated the rates of acetylcholine (ACh) synthesis and release in LA-N-2 cells in order to characterize them as a potential model of cholinergic neurons. When grown in a serum-containing medium the cells extend few neurites. In the absence of serum most cells develop processes. ACh content of the cells (determined by a radioenzymatic assay) varies with extracellular choline concentration in a saturable fashion, reaching a maximum of approximately 25 nmol/mg protein. Radiolabeled choline is taken up by the cell and converted to ACh or phosphocholine, as determined by purification from cell extracts by HPLC, in a saturable manner which is described by a single rectangular hyperbola. Hemicholinium-3 (100 microM) inhibits this uptake. The cells release ACh spontaneously and this release is enhanced upon depolarization with potassium or veratridine (the latter effect is blocked by tetrodotoxin). The data demonstrate that LA-N-2 cells exhibit some properties similar to cholinergic neurons and may therefore be useful for studies of ACh synthesis and release.