In this study neurite outgrowth from cultured hippocampal neurones was increased by addition of acetylcholinesterase acting in a non-cholinergic manner. Only monomeric acetylcholinesterase, a form of acetylcholinesterase dominant in development, increased neurite outgrowth (3-10 U/ml); moreover this effect was not blocked by active site blockers (echothiophate and galanthamine) but was sensitive to the addition of peripheral site blockers (fasciculin and BW284c51). It appears therefore that acetylcholinesterase has alternative, non-cholinergic functions, one of which could be in development, via a peripheral site. The possibility of a causal relationship between neurite outgrowth and calcium influx was explored using a spectrum of acetylcholinesterase variants, inhibitors and calcium channel blockers. Acetylcholinesterase regulation of outgrowth was shown to depend on an influx of extracellular calcium specifically via the L-type voltage-gated calcium channel. In summary, we propose that, independent of its catalytic activity, a selective form of acetylcholinesterase has a role in the development of hippocampal neurones via a selective voltage-gated calcium channel.