The intrinsic factors involved in the temperature-dependent impairment of neuronal activity in hippocampal CA2-CA1 regions were investigated using optical recording techniques. At 32°C, stimulation of the Schaffer collaterals in the hippocampal CA2 region evoked depolarizing optical responses that spread toward the CA1 region. The optical response was characterized by fast and slow components that were mainly related to the presynaptic action potentials and excitatory postsynaptic response, respectively. The increase of the temperature to 38°C was associated with a reversible depression of the neuronal activity in the hippocampal brain preparations. The depression of neuronal activity was irreversible when the temperature was increased to 40°C. In the presence of 22mM glucose, the depression of the neuronal activity at 38°C was significantly attenuated. Pyruvate (22mM), but not lactate (22mM), also improved the depression of neuronal activity induced by the temperature increase. Adenosine (200 μM) strongly depressed the excitatory postsynaptic response, but not presynaptic action potentials. 8-Cyclopentyl-1, 3-dimethylxanthine (8-CPT) (10 μM), an adenosine A₁ receptor blocker, attenuated the adenosine-induced depression of the excitatory postsynaptic response. 8-CPT (10 μM) prevented the impairment of the excitatory postsynaptic response induced by the increase of the temperature to 38°C. In contrast, the depression of presynaptic action potential at 38°C was not prevented by 8-CPT (10 μM). N_ω-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase (NOS) inhibitor, and methylcobalamin (10 μM), a vitamin B₁₂ analogue, attenuated the inhibition of pre- and postsynaptic activities induced by the increase of the temperature to 38°C Glibenclamide, a K_ATP channel blocker, did not protect neuronal activity from the effects of the increase of the temperature. These results suggest that the heat-induced depression of neuronal activity is mediated by multiple factors, such as impairment of energy metabolism and increase in extracellular adenosine and nitric oxide (NO) levels in hippocampal neurons.