Neurotransmitters have been reported to regulate neurite outgrowth in several vertebrate and nonvertebrate species. In this study, cultures of isolated embryonic day 12 (E12) chick sympathetic neurons were grown in the presence of cholinergic receptor agonists or antagonists. Both ACh and the nonhydrolyzable cholinergic agonist carbamylcholine (CCh) inhibited neurite outgrowth. ACh (0.1–1.0 mM) decreased the percentage of neurons bearing neurites, but had no significant effect on cell survival. The effect of ACh was increased in the presence of the cholinesterase inhibitors BW284C51 (1 microM), Tacrine (20 microM), and edrophonium (200 microM). Neurite outgrowth was strongly inhibited by the muscarinic receptor agonist oxotremorine (5–100 microM) and weakly inhibited by nicotine (50 nM to 10 microM). The inhibitory effect of CCh was decreased by the muscarinic receptor antagonist atropine (10 microM), demonstrating that the effect of CCh on neurite outgrowth was mediated, at least in part, through a muscarinic receptor. The possibility that AChE can influence neurite outgrowth directly, through a noncatalytic mechanism, was also examined. When dissociated chick brain or sympathetic neurons were grown on plates precoated with purified AChE, neurite outgrowth was strongly stimulated. However, the neurite outgrowth-promoting effect of AChE was strictly dependent upon the presence of substratum-bound heparan sulfate proteoglycans (HSPG). Pretreatment of AChE with diisopropylfluorophosphate to inhibit the esterase activity did not abolish this effect, suggesting that the neurite outgrowth-promoting effect of AChE was associated with a noncatalytic mechanism, a view supported by the observation that soluble AChE had no effect on neurite outgrowth.(ABSTRACT TRUNCATED AT 250 WORDS)