To investigate the effects of estrogen on the activity of the neurons in the hypothalamic ventromedial nucleus (VMN), the single-unit activity was recorded from in vitro brain tissue slices prepared from ovariectomized rats that were either treated or untreated with estrogen. In tissue slices continuously perifused with estrogen-free solution, half of the 348 VMN neurons studied were silent or nearly silent, and the rest fired spontaneously at low rates. Electrical stimulation of the periventricular region medial to the VMN evoked not only 3 types of orthodromic responses, but also antidromic responses from many VMN neurons, indicating that the VMN and the periventricular region are intimately and reciprocally connected. Neuronal responses to a battery of 6 neurotransmitters were studied by microinjecting 50 microliters of a transmitter solution directly into the chamber where the tissue slice was being continuously perifused. When effective, glutamate (Glut) was almost exclusively excitatory; acetylcholine (ACh) and norepinephrine (NE) could be excitatory and/or inhibitory; 5-hydroxytryptamine (5-HT) and dopamine (DA) were predominantly inhibitory; and gamma-amino-butyric acid (GABA) was exclusively inhibitory on neuronal activity. The overall resting neuronal activity and responses to the transmitter were very similar to those observed in in vivo studies, indicating that, for VMN at least, in vitro tissue slices can serve as a simplified model for studying neuronal activity and responses. Comparisons of results between estrogen-treated and untreated preparations showed that chronic, in vivo estrogen treatments, while having no effect on resting firing rate, selectively facilitated the responsiveness of silent (but not spontaneously active) neurons to excitatory orthodromic inputs, and the responsiveness to the excitatory action of ACh, the overall action of NE and the inhibitory action of 5-HT. These selective estrogen effects would no doubt alter the input-output relationships of VMN neurons evoked by the orthodromic inputs and ACh, NE and 5-HT. Since the VMN is crucial to many biological functions, and the cholinergic, adrenergic and serotonergic inputs to the ventromedial hypothalamus are involved in the estrogenic regulation of sexual behavior, reproductive neuroendocrine, and other biological functions, the alteration of the input-output relationships evoked by the orthodromic and transmitter inputs would affect the regulation of many functions.