We have used the patch clamp technique in combination with intracellular calcium measurements to measure simultaneously Ca 2+ entry and ionic currents activated by emptying of intracellular Ca 2+ stores (capacitative Ca 2+ entry and Ca 2+ release-activated Ca 2+ currents, CRAC) in human endothelial cells from umbilical veins. Intracellular stores were depleted of Ca 2+ by preincubating endothelial cells for 20 minutes with 2 μM thapsigargin in Ca 2+-free solution. Reapplication of 10 mM [Ca 2+] e evoked an increase in [Ca 2+] i indicating Ca 2+ influx after the thapsigargin-induced store depletion (capacitative Ca 2+ entry), however no measurable CRAC could be detected. The increase in [Ca 2+] i after [Ca 2+] e resubmission was substantially reduced in the presence of 50 μM NPPB (5-nitro-2-(3-phenylpropylamino)-benzoic acid) from 0.77 ± 0.25 μM to 0.2 ± 0.06 μM (n = 6) at a holding potential of −40 mV. Estimates of the capacitative Ca 2+ entry at various membrane potentials from the first time derivative of the Ca 2+ transients showed a highly inwardly rectifying I–V curve with a Ca 2+ inward current amplitude of 1.0 ± 0.3 pA (membrane capacitance 59 ± 9 pF, n = 8) at −80 mV. This current amplitude was decreased to 0.32 ± 0.12 pA( n = 6) in the presence of 50 μM NPPB. This corresponds to a decrease in the Ca 2+ permeability of the endothelial cell membrane from 0.15·10 −8 cm/s (control) to 0.06·10 −8 cm/s (50 μM NPPB).