Spatial and temporal calcium oscillations regulate multiple signalling pathways in endothelial cells and they are essential for proper endothelial cell function. When the normal pathways for interaction break down, as can occur in disease states, uncontrolled or asynchronous behaviour can occur. Increased levels of circulating RNA may result from excessive cell damage or cancer. We investigated the effect of double-stranded RNA (dsRNA) on calcium homeostasis, gene expression, trans-endothelial electric resistance and proliferation of human pulmonary artery endothelial cells (hPAECs). Cultured human PAECs from 10 donors were purchased from Lonza Basel, Switzerland and used in passages 4 to 8. Fura-2/am loaded hPAECs were used to investigate calcium changes of 24h incubation with Poly I:C (synthetic dsRNA), dsRNA, natural RNA or control solution by means of live-cell imaging. As a standard stimulus, 100µM histamine or 15µM BHQ (a selective inhibitor of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA)) was used in the presence and absence of extracellular calcium. Proliferation tests were performed on Poly I:C, dsRNA or BHQ stimulated cells, by means of 3H-thymidine incorporation. Gene expression of SERCA and toll-like-receptor 3 after dsRNA stimulation was analysed by quantitative RT-PCR. The cellular barrier properties of hPAECs as a read-out for cell function were assessed by measuring changes in the trans-endothelial electric resistance. The calcium response to histamine showed a significantly prolonged duration after dsRNA incubation (from 86 ± 3s to 131 ± 1s, n= 58 and n=77 respectively; p<0.01) but no increase in the basal level of calcium. There was no difference in the BHQ-induced Ca2+ response of the cells, pointing to an inhibitory effect of dsRNA on SERCA. Both BHQ and dsRNA inhibited hPAECs proliferation dose-dependently (IC50=60 ± 1 µM for BHQ and IC50=2.9 µg/ml for dsRNA). The quantitative RT-PCR showed that SERCA was 3-fold down-regulated by the dsRNA incubation. The electrical resistance of the monolayer was decreased by 50% after 24h dsRNA treatment (n=4). In conclusion, it is tempting to hypothesize that the inhibition and down-regulation of sarco/endoplasmic reticulum Ca2+-ATPase by double-stranded RNA in human pulmonary endothelial cells contributes to the endothelial dysfunction.