Rattlesnakes, copperheads, and other pit vipers have highly sensitive heat detectors known as pit organs, which are used to sense and strike at prey. However, it is not currently known how temperature change triggers cellular and molecular events that activate neurons supplying the pit organ. We dissociated and cultured neurons from the trigeminal ganglia (TG) innervating the pit organs of the Western Diamondback rattlesnake (Crotalus atrox) and the copperhead (Agkistrodon contortix) to investigate electrophysiological responses to thermal stimuli. Whole cell voltage-clamp recordings indicated that 75% of the TG neurons from C. atrox and 74% of the TG neurons from A. contortix showed a unique temperature-activated inward current (IDeltaT). We also found an IDeltaT-like current in 15% of TG neurons from the common garter snake, a species that does not have a specialized heat-sensing organ. A steep rise in the current-temperature relationship of IDeltaT started just below 18 degrees C, and cooling temperature-responsive TG neurons from 20 degrees C resulted in an outward current, suggesting that IDeltaT is on at relatively low temperatures. Ion substitution and Ca2+ imaging experiments indicated that IDeltaT is primarily a monovalent cation current. IDeltaT was not sensitive to capsaicin or amiloride, suggesting that the current did not show similar pharmacology to other mammalian heat-sensitive membrane proteins. Our findings indicate that a novel temperature-sensitive conductance with unique ion permeability and low-temperature threshold is expressed in TG neurons and may be involved in highly sensitive heat detection in snakes.