Abstract In order to characterize the neurons responsible for ACTH release and the suppression produced by adrenocortical steroids, we have studied the pharmacological sensitivity of neurons in the region containing the highest concentration of corticotropin releasing factor (CRF) in the brain and probably responsible for most of these activities. The effects of 8 putative neurotransmitter applied iontophoretically to more than 400 mediobasal hypothamamic (MBH) neurons in the rat were examined, and compared with cortisol sensitivity of the same neurons. Glutamate was the only agent that produced excitation exclusively, while GABA, serotonin, glycine and dopamine were inhibitory in action. Mixed excitation and inhibition were produced by histamine, acetylcholine (ACh) and norepinephrine (NE). All 8 cells excited by ACh were inhibited by cortisol and constitute only about 10% of neurons sampled; these neurons are considered to be strong candidates for CRF-releasing cells. On 7 of these neurons histamine was strongly excitatory and its action greatly outlasted the iontophoretic application. Histamine, however, excited many other neurons, including those neither excited by ACh nor steroids.