Spontaneous outward currents have been recorded in freshly isolated single vascular endothelial cells from the aorta. The currents reflect the activation of K⁺ channels, and are reminiscent of equivalent and well-characterized events in vascular smooth muscle. The latter serve to suppress muscle contraction, of particular importance in arteries developing spontaneous (myogenic) tone. Endothelial cells exert a significant predominantly inhibitory influence on vascular smooth muscle tone, which in small resistance arteries importantly involves a spread of hyperpolarization to the muscle. As the characteristics of vascular cells are known to change considerably as artery size decreases, we investigated whether endothelial cells in resistance-size arteries develop spontaneous outward currents. Endothelial cell sheets were isolated as previously described (McSherry I et al. 2005). Throughout all recording periods, the cells were continually superfused and kept at room temperature (21-23°C). For measurement of membrane currents, the conventional whole-cell patch-clamp technique was used. In most experiments membrane voltage was clamped at -20 mV, which is close to the resting membrane potential of these cells. In separate experiments, to measure changes in endothelial cell [Ca²⁺]_i, cells were incubated with the Ca²⁺ indicator Oregon Green 488 BAPTA-1 AM (10 µM) for at least 40 min. Fluorescence intensity (F) was recorded using a laser scanning confocal system (Olympus FV500) with Tiempo software at 0.5-1 Hz. Data were only used if the cell responded to 1 µM acetylcholine, and either evoked an outward current of at least 20 pA, or a clear increase in fluorescence intensity (F/F₀ greater than 0.3). Values are means ± S.E.M. Spontaneous outward currents were recorded from 10.3% of cells (n=58 sheets of cells). At a holding potential of -20 mV, outward current developed with a frequency of 0.09 ± 0.01 Hz (range 0.03-0.15 Hz) and amplitude of 107 ± 29 pA (n=6 sheets of cells). The amplitude of spontaneous currents decreased progressively as voltage was stepped from -20 mV to -70 mV (n=3 sheets of cells). Spontaneous oscillations in [Ca²⁺]_i were observed in 10.5% of cells (4 cells of 38 cells in 4 sheets of cells). The amplitude of spontaneous oscillations in [Ca²⁺]_i reached 69 ± 12% of the response to 1µM acetylcholine, and the frequency of [Ca²⁺]_i oscillations was 0.04 ± 0.02 Hz (n=4 cells). These data indicate that endothelial cells from small resistance arteries develop spontaneous outward currents, probably reflecting the activation of Ca²⁺-activated K⁺ channels. This suggests that spontaneous hyperpolarization originating in the endothelium may contribute to the regulation of vascular tone.