The regulation of neuromediator expression by neuronal activity in the enteric nervous system (ENS) is currently unknown. Using primary cultures of ENS derived from rat embryonic intestine, we have characterized the regulation of tyrosine hydroxylase (TH), a key enzyme involved in the synthesis of dopamine. Depolarization induced either by 40 mm KCl, veratridine or by electrical field stimulation produced a robust and significant increase in the proportion of TH immunoreactive (TH‐IR) neurons (total neuronal population was identified with PGP9.5 or Hu) compared to control. This increase in the proportion of TH‐IR neurons was significantly reduced by the sodium channel blocker tetrodotoxin (0.5 μm), demonstrating that neuronal activity was critically involved in the effects of these depolarizing stimuli. KCl also increased the proportion of VIP‐IR but not nNOS‐IR enteric neurons. The KCl‐induced increase in TH expression was partly reduced in the presence of the nicotinic receptor antagonist hexamethonium (100 μm), of noradrenaline (1 μm) and of the α₂‐adrenoreceptor agonist clonidine (1 μm). Combining pharmacological and calcium imaging studies, we have further shown that L‐type calcium channels were involved in the increase of TH expression induced by KCl. Finally, using specific inhibitors, we have shown that both protein kinases A and C as well as the extracellular signal‐regulated kinases were required for the increase in the proportion of TH‐IR neurons induced by KCl. These results are the first demonstration that TH phenotype of enteric neurons can be regulated by neuronal activity. They could also set the basis for the study of the pathways and mechanisms involved in the neurochemical plasticity observed both during ENS development and in inflammatory enteric neuropathies.