We examined the role played by intracellular Ca²⁺ stores in conducted vasomotor responses induced by phenylephrine (PE) in isolated hamster cremasteric arterioles. PE applied briefly (>1 sec) to isolated, cannulated arterioles using pressure-pulse ejection from a micropipette produced a strong local vasoconstriction and a very small biphasic conducted response (a small constriction followed by a dilation) that propagated several hundred microns along the vessel length. The conducted vasomotion was associated with a monophasic elevation of the endothelial cell [Ca²⁺]_i at the site of stimulation, as measured with the Ca²⁺ indicator fura-2. The Ca²⁺ pump inhibitor, thapsigargin, was used to limit filling of Ca²⁺ stores in smooth muscle and endothelial cells. Thapsigargin reduced baseline diameter, and elicited a strong dilator component at the local site, while enhancing both the constrictor and dilator components of the PE-induced conducted response. The enhanced conducted constrictor component induced by thapsigargin was mimicked by extraluminal application of tetraethylammonium (TEA) or charybdotoxin (CTX), but not by iberiotoxin (IbTX), apamin, glibenclamide, barium, or 4-aminopirydine (4-AP). Thapsigargin increased the estimated basal endothelial cell [Ca²⁺]_i by ~60 nM, and converted the PE-induced change in [Ca²⁺]_i from monotonic to biphasic with a late elevation of [Ca²⁺]_i above baseline that coincided with the increased dilatory component of the conducted response. Luminal application of CTX plus apamin significantly reduced the dilatory component of the conducted response. These results indicate that intracellular Ca²⁺ stores play a dynamic role in regulating conducted vasomotor responses, apparently through modulation of K_Ca channels in both cell types.