Anatomical evidence exists for projections to the tuberomammillary nucleus (TM) from the nucleus of the diagonal band of Broca (DBB) and the lateral preoptic area (LPO). The physiological effects of activating these inputs were studied by recording postsynaptic responses intracellularly from TM cells during both electrical stimulation and local nanodrop application of glutamate in horizontally cut brain slices. Electrical stimulation of the DBB, LPO and anterior lateral hypothalamic area (LH) usually evoked fast IPSPs (approximately 75% of responses) which were blocked by bicuculline or picrotoxin, suggesting GABA(A) mediation. The remaining excitatory responses evoked by stimulation of the LPO and LH were blocked by non-NMDA receptor antagonists (CNQX or NBQX) and the NMDA receptor antagonist, AP-5. Glutamate applied to the above areas induced postsynaptic responses in TM cells similar to those seen with electrical stimulation. Spontaneous firing in TM cells was suppressed by glutamate applied in the DBB. Glutamate applied in the LPO or LH evoked both inhibitory and excitatory responses. Changes in PSPs and firing rates were interpreted to result from glutamate activation of the neurons in the DBB, LPO and LH areas with inhibitory or excitatory connections to recorded TM neurons. These results support previous anatomical findings and suggest that inhibitory and excitatory synaptic control of TM activity is exerted by the DBB, LPO and LH areas.