Abstract Physiologic stressors increase trans-synaptic impulse activity and result in adrenal catecholamine release and biosynthesis. To determine the effects of stress on the co-localized opiate peptide system, rats were cold stressed at 4°C. While cold stress slightly decreased enkephalin levels, a more severe stress (wetting and cold) increased enkephalin levels by 95%. Examining trans-synaptic-cholinergic mechanisms, treatment with either nicotinic or muscarinic agonists alone resulted in no change in adrenal enkephalin content. However, treatment with both nicotinic and muscarinic agonists together resulted in a three-fold rise in enkephalin levels. To further examine cellular mechanisms, medullae were explanted in the presence of agents that increase second messenger cyclic nucleotide levels. Treatments that increase the levels of cAMP, the cyclic nucleotide associated with nicotinic receptor activation, prevented the rise in medullary enkephalin relative to control explants. In contrast, treatments that increased cGMP levels, the cyclic nucleotide associated with muscarinic receptor activation, had no effect on enkephalin content compared to control explants. However, in the presence of both forskolin (10 μM) plus db-cGMP (5 mM), enkephalin content rose three-fold over control explants. These data suggest that, distinct from catecholamine pathways, enkephalin levels can be positively or negatively regulated by the severity of a stressful stimulus, by cholinergic receptor mechanisms and by an interaction of cyclic nucleotide second-messenger pathways.