Over the past decade there has been great interest in the therapeutic potential of brain cooling for epilepsy, stroke, asphyxia and other neurological diseases. However, there is still no consensus regarding the neurophysiological effect(s) of brain cooling. We employed standard physiological techniques and 2-photon microscopy to directly examine the effect of temperature on evoked neurotransmitter release in rat hippocampal slices. We observed a monotonic decline in extracellular synaptic potentials and their initial slope over the temperature range 33–20°C, when the slices were cooled to a new set point in less than 5 s. Imaging the fluorescent synaptic marker FM1-43 with 2-photon microscopy showed that the same cooling protocol dramatically reduced transmitter release between 33 and 20°C. Cooling also reduced the terminal FM1-43 destaining that was induced by direct depolarization with elevated K+, indicating that axonal conduction block cannot account for our observations. The temperature dependence of FM1-43 destaining correlated well with the effect of temperature on field potential slope, compatible with a presynaptic explanation for our electrophysiological observations. Optical measurement of FM1-43 dissociation from cell membranes was not affected by temperature, and rapid cooling of slices loaded with FM1-43 did not increase their fluorescence. Our experiments provide visible evidence that a major neurophysiological effect of cooling in the mammalian brain is a reduction in the efficacy of neurotransmitter release. This presynaptic effect may account for some of the therapeutic benefits of cooling in epilepsy and possibly stroke.