AbstractThe effects of various neurotransmitter agonists and antagonists on the synthesis and release of methionine enkephalin (mENK) in neuronal cultures of mouse spinal cord and dorsal root ganglia have been measured. Blockade of electrical activity with tetrodotoxin between days 9 and 13 in culture caused a>95% decrease in the number of mENK‐immunoreactive neurons. This effect was also seen upon the blockade of glycine and β‐adrenergic receptors with strychnine and propranolol, respectively, and stimulation of GABA receptors with muscimol. Stimulation of β‐adrenergic receptors with isoproterenol, or blockade of glutamate and GABA receptors with 2‐aminophosphonovalerate and strychnine, respectively, had a qualitatively opposite action on both the number of mENK‐immunoreactive neurons and enkephalin peptide levels measured by radioimmunoassay. Application of substance P also enhanced the mENK cell number. These data suggest that, at least in the spinal cord, characteristics other than the average level of impulse activity in the afferent input may be critical to the regulation of express