We investigated long-lasting interactions that may occur between two forms of intercellular signaling: cholinergic synaptic transmission and gap junction-mediated coupling in the rat adrenal medulla. The junctional coupling between chromaffin cells was studied during reduced or blocked synaptic transmission in adrenal slices. First, cholinergic synaptic activity was reduced by pharmacological treatment. Bath-application of the nicotinic receptor antagonists hexamethonium, the oxystilbene derivative F3, or alpha-bungarotoxin, acting at distinct neuronal-like postsynaptic nicotinic acetylcholine receptors (nAChRs), significantly increased the incidence of Lucifer yellow passage (dye coupling) between chromaffin cells (p > 0.7 in treated slices vs p = 0.4 in controls). Dye coupling was associated with an elevated macroscopic conductance of the junctional current measured by dual patch-clamp. Pharmacological inhibition of protein trafficking from the trans-Golgi network to the plasma membrane by either brefeldin A or nocodazole pretreatment prevented the effects of nAChR antagonists on dye coupling. Interestingly, this upregulation of gap junction-mediated coupling in response to reduced synaptic activity is of physiological relevance, because it is found in the newborn rat, in which cholinergic synaptic transmission has not yet matured. This mechanism may also be of importance in pathological conditions, because chronic blockade of synaptic transmission after surgical denervation of the adrenal gland also resulted in increased dye coupling between chromaffin cells. In conclusion, our pharmacological, physiological, and pathological data concur to demonstrate that gap junction-mediated intercellular communication between chromaffin cells undergoes persistent adaptation in response to impairment of synaptic activity. These results strongly suggest that gap junctional communication between chromaffin cells is under tonic inhibitory control exerted by cholinergic synaptic inputs.