The T-type Ca²⁺ channel Ca_V3.1 subunit is present in pulmonary microvascular endothelial cells (PMVECs), but not in pulmonary artery endothelial cells (PAECs). The present study sought to assess the role of Ca_V3.1 in thrombin-induced Weibel-Palade body (WPB) exocytosis and consequent von Willebrand factor (VWF) release. In PMVECs and PAECs transduced with a GFP-tagged VWF chimera, we examined the real time dynamics and secretory process of VWF-GFP-containing vesicles in response to thrombin and the cAMP-elevating agent isoproterenol. While thrombin stimulated a progressive decrease in the number of VWF-GFP-containing vesicles in both cell types, isoproterenol only decreased the number of VWF-GFP-containing vesicles in PAECs. In PMVECs, thrombin-induced decrease in number of VWF-GFP-containing vesicles was nearly abolished by T-type Ca²⁺ channel blocker mibefradil as well as by Ca_V3.1 gene silencing with small hairpin RNA (shRNA). Expression of recombinant Ca_V3.1 subunit in PAECs resulted in pronounced increase in thrombin-stimulated Ca²⁺ entry, which is sensitive to mibefradil. Taken together, these data indicate that VWF secretion from lung endothelial cells is regulated by two distinct pathways involving Ca²⁺ or cAMP, and support the hypothesis that activation of Ca_V3.1 T-type Ca²⁺ channels in PMVECs provides a unique cytosolic Ca²⁺ source important for G_q-linked agonist-induced VWF release.