The endothelium has been shown to contribute to the modulation of vascular smooth muscle tone. Using digital imaging microscopy, we measured calcium in endothelial cells of isolated skeletal muscle arterioles. First-order arterioles (N = 6) were isolated from rat cremaster muscle and cannulated. All vessels developed spontaneous tone (99.4 microns; 60%-80% of passive diameter). Endothelial cells were loaded with Fura-2-AM (5 microM), a ratiometric calcium-sensitive fluorescent dye. The Fura-2-AM was placed in the lumen of the vessel for approximately 10 min and removed. Fluorescent images of endothelial cells were acquired following excitation at 340- and 380-nm wavelengths and the data expressed as the 340/380 ratio which is directly proportional to intracellular calcium. Acetylcholine (Ach; 10(-6) M), an endothelium-dependent dilator, caused significant dilation (132% of control) and increased calcium to 186% of control. Adenosine (Adn; 10(-4) M), an endothelium-independent agent, produced similar dilation (127% of control) but did not alter endothelial cell calcium. Increasing intraluminal pressure produced a myogenic constriction with no change in endothelial calcium. Flow, induced by a 20 cm H2O pressure gradient, failed to dilate the arterioles and produced no increase in endothelial cell calcium. However, flow induced by a 40 cm H2O pressure difference did increase endothelial cell calcium prior to a significant arteriolar dilation. Removal of the endothelium by physically rubbing the intimal surface eliminated both the dilation and increase in calcium to Ach or flow (40 cm H2O) while the response to Adn remained unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)