We measured endothelial Ca2+ concentration ([Ca2+]) in juxtamedullary afferent arterioles in response to step changes in perfusion pressure. Measurements were made with fura 2 using a fluorescence-imaging system designed to measure Ca2+ in whole tissues and minimize potentially harmful effects of ultraviolet illumination. The system yielded the typical sigmoidal relationship between fluorescence-emission ratio and [Ca2+] in vitro and was sensitive to endothelial Ca2+ transients elicited by bradykinin. Our goal was to determine whether changes in endothelial Ca2+ trigger events that cause myogenic vasoconstriction. Bradykinin, acting via endothelial cells, and sodium nitroprusside (SNP), acting independently, triggered vasodilation; bradykinin but not SNP increased endothelial Ca2+. Increased perfusion pressure caused vasoconstriction and a modest rise in endothelial Ca2+. Because the rise in Ca2+ with bradykinin initiates the vasodilation, the small rise in Ca2+ with pressure cannot cause vasoconstriction. Our results suggest that myogenic constriction is triggered from within vascular smooth muscle cells and that some other phenomenon, most likely increased shear stress, increases endothelial Ca2+ and modulates the myogenic reaction.