The use of single- and dual-wavelength Ca(2+)-sensitive fluorescent dyes to monitor changes in endothelial and/or smooth muscle intracellular Ca(2+) levels has provided information linking Ca(2+) events to changes in arterial function. Here we describe the in vitro techniques used to selectively load Ca(2+) indicators into either the endothelium or the smooth muscle of cannulated rat cremaster arteries. These vessels normally develop spontaneous myogenic tone that is largely unaffected by the loading of Ca2+ indicators or the subsequent imaging procedures. This suggests that there is minimal Ca2+ buffering or damage, and that the fluorescent indicator-loaded vessels behave similarly to unloaded preparations. Importantly, these approaches are applicable to both isobaric and isometric preparations and have been also used for the study of a number of vascular beds including cerebral, mesenteric, coronary, and skeletal muscle vasculatures.