Insulin may decrease the contractile response of vascular smooth muscle to vasoactive agents. This could be due to interactions of insulin with the effects of vasoactive agonists on intracellular free calcium transients in vascular smooth muscle cells. This study assesses the effects of physiological doses of insulin (70 μU/mL) on calcium responses in cultured vascular smooth muscle cells (primary unpassaged and passaged) to angiotensin II (1 nmol/L), arginine vasopressin (10 nmol/L), and norepinephrine (10 μL). Intracellular free Ca2± concentrations in single cells were measured microphotometrically using fura 2-AM. Insulin, angiotensin II, arginine vasopressin, and norepinephrine significantly increased calcium (to 115±7, 183±20, 184±15, and 168±12 nmol/L, respectively, from basal calcium of 90±10 nmol/L). Insulin si gnificantly attenuated the agonist-induced calcium responses. The effects of insulin were almost completely inhibited by diltiazem, staurosporine, calphostin C, and thapsigargin. In conclusion, insulin stimulates calcium transients but blunts agonist-mediated calcium rises in vascular smooth muscle cells. These responses are related to regulatory effects of insulin on cellular calcium homeostasis and may explain how insulin modulates vascular smooth muscle contraction.