Fura 2 microfluorometry and perforated-patch whole cell recording were carried out simultaneously to investigate the relationship between intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and membrane current activation in response to ACh and caffeine in freshly dissociated arterial endothelial cells. ACh and caffeine evoked transient increases in [Ca²⁺]_i. The initial increase in [Ca²⁺]_i was accompanied by a transient outward current, which caused membrane hyperpolarization. The amplitudes of the [Ca²⁺]_i transient and outward current were dependent on caffeine concentration (EC₅₀ ~ 1 mM). Cyclopiazonic acid raised resting [Ca²⁺]_i levels by 50 nM and failed to completely block caffeine- or ACh-induced [Ca²⁺]_i transients but slowed [Ca²⁺]_i recovery fourfold. The reversal potential of caffeine-induced currents was dependent on external K⁺ and Cl concentrations. Caffeine-induced current amplitudes, but not [Ca²⁺]_i responses, were attenuated by external tetraethylammonium, Zn²⁺, and La³⁺. A consistent temporal relationship between agonist-activated membrane current and [Ca²⁺]_i increases was not observed, and, in some cases, time differences were greater than expected for simple diffusion of Ca²⁺ throughout the cell. These results suggest that Ca²⁺-dependent current activation monitors local [Ca²⁺]_i changes adjacent to the plasmalemma, whereas single-cell photometry provides a measure of global changes in [Ca²⁺]_i.