The morphogenetic role of the neurotransmitter acetylcholine was studied in cultures of dorsal root ganglia (DRG) neurons obtained from E12 and E18 chick embryos. With this model we have evaluated neurofilament expression and neurite outgrowth following cholinergic agonist and antagonist treatment. Morphometric analysis undertaken to evaluate fiber outgrowth has indicated that E12 DRG cultures treated with cholinergic agonists, such as muscarine and carbachol, when compared with untreated cultures, have longer fibers and a higher number of fibers per neuron. Concomitant treatment with agonists and the antagonists atropine or mecamylamine counteracts the increase in fiber outgrowth, suggesting that the cholinergic agonist effects were mediated by both muscarinic and nicotinic receptors. The expression of the three neurofilament proteins was also evaluated. Western blot analysis showed that, in E12 DRG cultures, both muscarine and carbachol induce a significant increase in neurofilament protein expression and that this effect is inhibited by cholinergic antagonist treatment. Moreover, Northern blot analysis has demonstrated that the increased expression of 68‐ and 145‐kDa neurofilament proteins is dependent on cholinergic modulation of the neurofilament transcripts. Modulated expression of neurofilament proteins by cholinergic agonists was not evident in E18 DRG cultures, suggesting that, when sensory neurons have completed their differentiation, the cholinergic system might be involved in other functions. In conclusion, our data demonstrate that, during sensory neuron development, acetylcholine modulates neurite outgrowth controlling neurospecific marker expression. © 2003 Wiley‐Liss, Inc.