We have measured the effects of thapsigargin, a specific inhibitor of endoplasmic CaZ+-adenosine 5'triphosphatase (CaZ+-ATPase), on membrane currents and on the intracellular Ca 2+ concentration ([Ca2+]i) in single endothelial cells from the human umbilical cord vein. Currents were recorded by means of the patchclamp technique in the whole-cell mode and [Ca 2 +]i was measured using Fura II. Application of thapsigargin at concentrations between 0.2 and 2 gmol/1 induced a slow increase in [Ca2+]i to a peak value of 400 + 110 nmol/1 above a resting level of 120 _+ 35 nmol/1, and then slowly declined to a new steady-state level of 315 _4-90 nmol/1 (n = 33). The thapsigargin-induced increase in [Ca 2 +]i depended on the extracellular Ca 2 + concentration ([Ca 2 +]o: it declined after removal of extracellular Ca 2 +, but increased again when [Ca2+]o was augmented, indicating that the response depends on a transmembrane influx of Ca 2 + ions. The peak amplitude of the histamine-induced Ca 2+ transient was reduced in the presence of thapsigargin. This reduction was more pronounced when histamine was appfied at the peak of the increase in [Ca 2 +]i induced by thapsigargin than during the rising phase of the changes in [Ca 2 +]i. The decline of the Ca 2 + transient induced by histamine after washing out the agonist was also affected by thapsigargin. Before application of thapsigargin, this decline could be described by a single exponential with a time constant z equal to 24.5 -t= 5 s (n = 7). In the presence of thapsigargin, the decline was much slower (n = 8 cells), although in four cells a fraction of about 23% still exchanged with a similar fast z value of 29.4 +_ 4 s. Thapsigargin also induced a slowly developing inward current in endothelial cells at a holding potential of -40 mV. Voltage ramps applied before and during the development of this current indicated that a nonselective cation channel with a reversal potential near 0 mV was activated. In contrast with the Ca 2 + transients, these currents did not show a declining phase. These results indicate that inhibition of the endoplasmic Ca 2 + Correspondence to. B. Nilius pump in endothelial cells increases [Ca2+]i. The tonic component of this increase might be partly due to opening of non-selective Ca z +-permeable cation channels activated by depletion of intracellular stores.