The enteric nervous system is derived from neural crest cells that emigrate from the hindbrain, enter the foregut and colonise the entire length of the gastrointestinal tract. Previous studies have shown that although enteric neural crest-derived cells migrate in chains, they have the ability to detach from their existing chain in order to join or form a new chain. In this study, the possible role of matrix metalloproteinase-3, -8 and -2/-9 on the migration of enteric neural crest-derived cells and the formation of the neural network within the developing gut were examined using specific pharmacological inhibitors. Blocking MMP-2/MMP-9 activity significantly decreased the distance that enteric neural crest-derived cells migrated through the developing gut. Morevover, the reticulated network formed by these cells was less complex. MMP-3 and MMP-8 inhibitors had no effect on neural crest migration. Expression studies showed that MMP-2, but not MMP-9, was expressed within the developing mouse gut. Collectively, the data suggest that MMP-2 activity is important for enteric neural crest-derived cell migration and the formation of the neural crest network.