In endothelial cells, agonist-induced Ca2+ entry takes place via the store-operated Ca2+ entry pathway and/or via channel(s) gated by second messengers. As cell stimulation leads to both a partial Ca2+ store depletion as well as the production of second messengers, these two pathways are problematic to distinguish. We showed that passive endoplasmic reticulum (ER) depletion by thapsigargin or cell stimulation by histamine activated a similar Ca2+-release-activated Ca2+ current (CRAC)-like current when 10 mM Ba2+/2 mM Ca2+ was present in the extracellular solution. Importantly, during voltage clamp recordings, histamine stimulation largely depleted the ER Ca2+ store, explaining the activation of a CRAC-like current (due to store depletion) upon histamine in Ba2+ medium. On the contrary, in the presence of 10 mM Ca2+, the ER Ca2+ content remained elevated, and histamine induced an outward rectifying current that was inhibited by Ni2+ and KB-R7943, two blockers of the Na+/Ca2+ exchanger (NCX). Both blockers also reduced histamine-induced cytosolic Ca2+ elevation. In addition, removing extracellular Na+ increased the current amplitude which is in line with a current supported by the NCX. These data are consistent with the involvement of the NCX working in reverse mode (Na+ out/Ca2+ in) during agonist-induced Ca2+ entry in endothelial cells.