The effect of Evans blue (EB) on large-conductance Ca 2 + -activated K + (BK C a ) channels was investigated in cultured endothelial cells of human umbilical veins. In whole-cell configuration, EB (50 μM) reversibly increased the amplitude of K + outward currents (I K ). When the patch pipettes were filled with 10 mM EGTA, its stimulatory effect onI K was unaltered. Further application of EB in the presence of suramin, a blocker of P 2 -purinergic receptor, or AOPCP, an inhibitor of 5'-nucleotidase, still increasedI K . However, charybdotoxin (100 nM) suppressed EB-induced increase inI K . In inside-out configuration, bath application of EB (50 μM) did not change single channel conductance but significantly increased the activity of BK C a channels. The EB-induced increase in the activity of BK C a channels was independent on internal Ca 2 + . EB (50 μM) shifted the activation curve of BK C a channels to less positive membrane potentials by approximately 20 mV. The change in the kinetic behavior of BK C a channels caused by EB in these cells is due to an increase in mean open time and a decrease in mean closed time. These results indicate that EB can stimulate the activity of BK C a channel in endothelial cells. This effect is unrelated to its blockade of P 2 -purinergic receptors or inhibition of 5'-nucleotidase. The direct stimulation of these ionic channels by EB may contribute to its effect on capillary permeability.