TRPM8 is a polymodal cation channel found in sensory neurons and other tissues. Its main agonists are low temperatures (<26°C) and natural compounds like menthol. TRPM8 is also modulated by many signaling pathways which can change its activity. Thus, we studied tyrosine phosphorylation of TRPM8, an important process in cell signaling that had not been investigated for this particular receptor-channel. HEK293T cells were co-transfected with human TRPM8 which had a VSV-G tag (VSV-G-TRPM8) and Src kinase (SrcY527F). After 48h, cells were treated with PP2 (10µM), a selective Src kinase inhibitor. VSV-G-TRPM8 was immunoprecipitated from cell lysates with VSV-G antibody and its phosphorylation was analyzed using the Western blot assay. VSV-G-TRPM8 is phosphorylated in cells expressing SrcY527F and PP2 treatment prevented TRPM8 phosphorylation (3 replicates). HEK293T cells stably transfected with human TRPM8 (HEK293T-hTRPM8) were transfected with siRNA to reduce endogenous Src expression (Src_siRNA) and a scrambled RNA was used as control. A Western blot assay was performed to verify Src expression. Src kinase expression was reduced after 96h in Src_siRNA-transfected cells, compared to scrambled RNA transfection (2 replicates). Calcium microfluorimetry was used to monitor the acute effect of PP2 on cold-induced calcium transients in HEK293T-hTRPM8 cells. Cold ramps (from ~32 to ~25°C) were applied seven times at 5 min interval, in the presence and in the absence of PP2 (10µM). Values represent mean±S.E.M. compared by Student’s t-test. Tachyphylaxis of cold-induced calcium transients occurred in the presence of PP2 in HEK293T-hTRPM8 cells transfected with both Src_siRNA and scrambled RNA but the effect was significantly more pronounced in scrambled RNA-transfected cells. The ratio between the amplitudes of the 7th and the 1st cold-induced calcium transients was 0.73±0.01 in scrambled RNA-transfected cells (n=336) and 0.90±0.01 in Src_siRNA-transfected cells (n=335), p<0.01. Adult Wistar rats (male, n=3) were used. Rats were killed by inhalation of 100% CO2 followed by decapitation. Dorsal root ganglia (DRG) were removed and prepared for the primary culture. Acute PP2 treatment (20 min) also partly inhibited cold-induced calcium transients in menthol-sensitive DRG neurons, from 0.56±0.06 to 0.37±0.06, n=27, p<0.05 (Fig.1). In the absence of PP2 there was no significant change in the amplitude of cold-induced calcium transients (0.64±0.07 during the first cold ramp, compared to 0.61±0.06 during the 3rd cold ramp, n=24). These results indicate that TRPM8 is tyrosine phosphorylated by Src kinase, as PP2 treatment prevents TRPM8 phosphorylation. The calcium microfluorimetry data suggest a positive modulation of the Src protein kinase on the TRPM8 channel, the phosphorylation of TRPM8 potentiating its response to moderate cooling. It is important that TRPM8 activity is reduced by PP2 treatment in neurons, where both TRPM8 and Src are expressed natively.