Two cold-sensitive ion channels have been cloned recently, both belonging to the transient receptor potential (TRP) channel family. TRPM8 is menthol sensitive and is activated by gentle cooling (McKemy et al., 2002; Peier et al., 2002). ANKTM1 (or TRPA1) is menthol insensitive and requires stronger cooling for activation (Story et al., 2003). The super-cooling agent icilin activates both channels. Using calcium imaging in trigeminal ganglion neurones two types of cold responses were recently described based on temperature thresholds (Thut et al., 2003). However, the molecules underlying the two types of responses are not yet known.We have used [Ca²⁺]_i imaging on rat DRG neurones loaded with Calcium Green-1 (Reid et al., 2002) to classify cold responses based on the known pharmacology of cold-sensitive ion channels. Wistar rats were humanely killed by CO₂ inhalation followed by decapitation. Cells which responded to a cooling ramp from 36 °C to 14 °C with a fluorescence increase over its initial level (∆F/F₀) higher than 0.15 were considered cold-sensitive.Of a total of 579 neurones, 136 were cold-sensitive. Based on the effect of 100 µM menthol, these cells were classified as menthol-sensitive (MS, 92 cells) or menthol-insensitive (MI, 44 cells). Neurones were considered MS if they responded to menthol at 32 °C or their cold response was sensitised by menthol. Fluorescence signals during cooling (∆F/F₀ = 0.41 ± 0.27 compared to 0.24 ± 0.1, mean ± SD, p < 0.001, Student’s t test) and temperature thresholds (25.3 ± 3.9 °C compared to 22.9 ± 2.7 °C, p < 0.001) were significantly higher for the MS group.Icilin sensitivity (50 µM at 32 °C) was restricted almost exclusively to MS cells (22/92 compared to 2/44 for the MI group, p = 0.002, χ² test) and significantly more MS than MI cells were sensitive to 2 µM capsaicin at 32 °C (61/92 compared to 22/44, p = 0.02, χ² test).In terms of separating cold responses into homogeneous groups, menthol sensitivity is a very efficient parameter: temperature thresholds, amplitudes of calcium signals and icilin sensitivity all follow the classification imposed by menthol sensitivity. The MS group is more sensitive to cooling and icilin and it is likely to express TRPM8. We have found no evidence that ANKTM1 is responsible for cold sensitivity in the MI group. All procedures accord with the principles of UK legislation.