Abstract The release of enzymatically active acetylcholinesterase was measured from two types of neuronal cell cultures. Evidence is presented that primary cultures of chick optic lobe and clonal rat PC 12 cells released acetylcholinesterase by similar mechanisms. The secretion was selective for the tetrameric or larger molecule forms of acetylcholinesterase. Thus the chick primary cultures released the so-called G4 isoenzyme of acetylcholinesterase. The rat PC 12 cells, after pretreatment with nerve growth factor, secreted mainly G4 acetylcholinesterase and a small amount of an isoenzyme sedimenting at 16S. The rate of secretion of acetylcholinesterase was regulated by the three types of agents used. (1) Acetylcholinesterase secretion was markedly increased by nerve growth factor. (2) Microtubule inhibitors (colchicine, taxol and nocodazole) suppressed the rate of release of acetylcholinesterase. (3) Acetylcholinesterase release was also decreased by an inhibitor of protein glycosylation, tunicamycin. We provide evidence that these three types of agents regulate acetylcholinesterase secretion not by a direct effect on secretion, but by modulatory effects on the synthesis, intracellular transport and turnover of the enzyme. (1) Nerve growth factor acts on acetylcholinesterase secretion via increased synthesis of messenger RNA. (2) Microtubule inhibitors act by preventing intracellular transport. (3) Tunicamycin acts by decreasing the intracellular activity of acetylcholinesterase, possibly via enhanced proteolytic breakdown.