The responses of neurons in the arcuate nucleus (ARC) to gamma-aminobutyric acid (GABA), glycine, serotonin (5-HT), norepinephrine (NE), and dopamine (DA) were examined in hypothalamic slices from ovariectomized rats either treated or untreated with estrogen. In estrogen-untreated preparations, spontaneous unit activity of the majority of neurons identified antidromically by the median eminence (ME) stimulation was depressed by GABA and glycine. When effective, 5-HT was predominantly excitatory and DA was predominantly inhibitory, while NE evoked both excitatory and inhibitory responses. The IS-SD block in evoked spikes was also occasionally observed following GABA and glycine perfusion. The responsiveness of unidentified neurons, which were not activated by the ME stimulation, to agents other than 5-HT did not differ from that of antidromically identified neurons. Responses of unidentified neurons to 5-HT were predominantly inhibitory. Subcutaneous injection of 20 micrograms of 17 beta-estradiol-benzoate 2 days prior to the experiments did not significantly affect the responsiveness of both identified and unidentified neurons to GABA, 5-HT, DA, and NE. Contrarily, preincubation of slices in 17 beta-estradiol-containing medium (10 nM) for 1 h significantly increased the responsiveness of unidentified, but not identified, neurons to the inhibitory action of NE. These results suggest that GABA, glycine, 5-HT, NE, and DA play some functional roles in controlling the excitability of tuberoinfundibular (TI) neurons in the ARC. It is also suggested that the negative feedback effect of estrogen is mediated, at least in part, by increased responsiveness of ARC neurons other than TI neurons to the inhibitory action of NE.