Introduction: Levels of the potent vasoconstrictor, endothelin-1 (ET-1), are elevated in several pathological states, including atherosclerosis (Ivey et al., 2008), vasospasm (Clark and Pyne-Geithman, 2005), pulmonary hypertension (Barman, 2007) and coronary slow flow phenomenon. Vasoconstriction is mediated by the activation of voltage-gated calcium channels (VGCC) and intracellular sarcoplasmic reticulum (SR) calcium release, which elevates intracellular cytosolic calcium concentration. Inhibition of myosin phosphatase by protein kinase C/CPI-17 or the RhoA/Rho kinase-dependent pathways enhances calcium sensitisation, further augmenting vasoconstriction. However, recent evidence has also implicated the involvement of non-selective cation channels (NSCC) in the activation of VGCC (Wang et al., 2008). Aims: This study aims to (1) document the role of SR calcium and calcium sensitisation in ET-1-mediated vasoconstriction, (2) identify the extent to which L-type calcium channel blockade attenuates extracellular calcium entry and vasoconstriction, and (3) identify whether NSCC are important in mediating activation of L-type calcium channels in ET-1-mediated vasoconstriction. Methods and results: Male Sprague-Dawley rats, weighing 350g were killed by carbon dioxide inhalation. Primary smooth muscle cells from rat mesenteric arteries coupled with patch clamping analysis demonstrated that ET-1 (100nM) increased the amplitude of L-type calcium current (n=9). Using functional wire myography, pharmacological blockade of VGCC using nifedipine (10µM) significantly attenuated the ET-1-mediated vasoconstriction (p<0.05; n=9). Depletion of SR calcium using cyclopiazonic acid (10µM) and the IP3 receptor and NSCC blocker, 2-APB (100µM), also reduced ET-1-dependent vasoconstriction (p<0.05; n=9). Ionic substitution of Na+ with NMDG+ did not alter the magnitude or rate of ET-1 developed tension, suggesting that Na+ entry through NSCC was not important in ET-1-dependent contraction (p>0.05; n=4-5). Using an EGTA/ calcium-free solution to deplete both extra and intracellular calcium stores revealed the involvement of calcium sensitisation pathways (p<0.05; n=8) and was consistent with biochemical analysis, documenting an increase in the Thr855 phosphorylation state of myosin phosphatase (p<0.05; n=8). All data were expressed as means ± SEM and analysed using Students paired or unpaired t-test, as appropriate. Comparisons between treatment groups were analysed using one-way analysis of variance (ANOVA) followed by Bonferroni correction. Conclusion: These data highlighted the therapeutic potential of SR calcium blockade but not NSCC blockade in mediating ET-1-dependent contraction.