The non-selective cation channel TRPM8 is present and functional on vascular smooth muscle (Yang et al., 2006; Johnson et al., 2009). The TRPM8 agonist menthol causes vasoconstriction in relaxed vessels or vasodilatation in precontracted vessels (Johnson et al., 2009). These diverse actions suggest multiple actions of this and other TRPM8 agonists. Previous studies have revealed that menthol can potently inhibit voltage-gated Ca2+ currents (ICa) in DRG and cardiac muscle cells (Swandulla et al., 1987; Baylie et al., 2010). In the present study we investigated whether menthol and other TRPM8 agonists may have actions in vascular smooth muscle mediated by such an additional mechanism. Tail arteries were obtained from humanely-dispatched male Sprague-Dawley rats (12 weeks). Single vascular smooth muscle cells (VSMC) were isolated by enzymatic digestion for patch clamp analysis. Vascular rings were endothelium denuded for isometric contraction studies. In voltage-clamped tail artery VSMCs, TRPM8 agonists icilin (50 μM), menthol (300 μM) and WS-12 (50 μM) were found to significantly reduce peak ICa compared to their respective vehicle-only controls (one-way ANOVA, P<0.001; n=8; N=3, where N = number of rats, n = number of cells). Isometric contraction studies demonstrated that menthol (300 μM) caused vessels precontracted with phenylephrine (PE: 2 μM; 0.71±0.06 g (mean ± SEM), N=15, n=19, where N= number of rats, n = number of preparations) to initially contract (108.4±7.3 % of PE control, P<0.01, one-way ANOVA, n=26, N=19) and then relax (73.5±2.4 % of PE control, P<0.001). However, when vessels were pre-incubated with L-type calcium antagonist, nifedipine (10 μM), PE-induced contractions were decreased by 46.5±3.0 % (n=41, N=15) compared to control conditions and the major response to menthol application was a strong contraction (140±8 % of control, P<0.001, N=7, n=10). When vessels were incubated with the TRPM8 antagonist, AMTB (10 μM), this menthol-induced contraction was strongly inhibited (TRPM8: 80±9 % of control, P<0.001, N=9, n=9). We conclude that the vasodilatatory actions of TRPM8 agonist, menthol, on precontracted vascular smooth muscle is likely to reflect effective L-type calcium channel blockade. Without this additional effect of menthol on calcium channels (e.g. in the presence of nifedipine), a TRPM8-specific contraction is revealed.