Gap junctions have traditionally been described as transmembrane channels that facilitate intercellular communication via the passage of small molecules. Connexins, the basic building blocks of gap junctions, are expressed in most mammalian tissues including the developing and adult central nervous system. During brain development, connexins are temporally and spatially regulated suggesting they play an important role in the proper formation of the central nervous system. In the current study, connexins 32 and 43 were overexpressed in PC12 cells to determine whether connexins are involved in neuronal differentiation. Both connexin 32 and 43 were appropriately trafficked to the cell membrane following overexpression and resulted in the formation of functional gap junctions. Connexin overexpression was found to cause enhanced neurite outgrowth in PC12 cells treated with nerve growth factor to initiate neuritogenesis. Surprisingly, however, enhanced neurite outgrowth was found to be the consequence of functional hemichannel formation as opposed to traditional intercellular communication. Additional analysis revealed that ATP was released into the media likely through hemichannels and acted on purinergic receptors to cause enhanced neurite outgrowth. Collectively, the results of the current study suggest that connexins may play an important role in neuronal differentiation by non-traditional mechanisms.