Changes in temperature of up to several degrees have been reported in different brain regions during various behaviors or in response to environmental stimuli. We investigated temperature sensitivity of dopaminergic neurons of the rat substantia nigra pars compacta (SNc), an area important for motor and emotional control, using a combination of electrophysiological techniques, microfluorometry and RT-PCR in brain slices. Spontaneous neuron firing, cell membrane potential/currents and intracellular Ca²⁺ level ([Ca²⁺]_i) were measured during cooling by up to 10° and warming by up to 5° from 34°C. Cooling evoked slowing of firing, cell membrane hyperpolarization, increase in cell input resistance, an outward current under voltage- clamp, and a decrease of [Ca²⁺]_i. Warming induced an increase in firing frequency, a decrease in input resistance, an inward current, and a rise in [Ca²⁺]_i. The cooling-induced current, which reversed in polarity between -5 and -17 mV, was dependent on extracellular Na⁺. Cooling-induced whole-cell currents and changes in [Ca²⁺]_i were attenuated by 79% in the presence of 2-aminoethoxydiphenylborane (2-APB; 200 µM), and the outward current was reduced by 20% with ruthenium red (100 µM). RT-PCR conducted with tissue punches containing the SNc revealed mRNA expression for TRPV3 and TRPV4 channels, known to be activated in expression systems by temperature changes within the physiological range. 2-APB, a TRPV3 modulator, increased baseline [Ca²⁺]_i, while 4-PDD, a TRPV4 agonist, increased spontaneous firing in 7 of 14 neurons tested. We conclude that temperature-gated TRPV3 and TRPV4 cationic channels are expressed in nigral dopaminergic neurons and are constitutively active in brain slices at near physiological temperatures, where they affect the excitability and calcium homeostasis of these neurons.