The purinergic signalling in the human blood-brain barrier endothelial cell line hCMEC/D3 was characterised by molecular biological, gene silencing, and Ca²⁺ imaging techniques. RT-PCR experiments showed the mRNA expression of the G-protein coupled receptor subtypes P2Y₂, P2Y₆, and P2Y₁₁, as well as the ionotropic P2X₄, P2X₅, and P2X₇ receptor subtypes. The application of the purinergic receptor subtype agonists ATP, ATPgS, BzATP, UDP, UTP, and ab-meATP revealed that the cells react to purinergic stimulation by a Ca²⁺ signal. Inhibitors of intracellular mediators (U73122 and 2-APB) antagonised the agonist induced Ca²⁺ signal, suggesting a G-protein coupled signalling pathway, probable coupled to the observed metabotropic P2Y-receptor subtypes P2Y₂, P2Y₆, and P2Y₁₁. The siRNA mediated gene silencing of the P2Y₂ receptor subtypes suppressed the possibility to induce a Ca²⁺ signal by the used agonists - even the P2Y₆ and P2Y₁₁ more specific agonists UDP and BzATP or ab-meATP were not able to induce the previously observed Ca²⁺ signals. An outstanding role of the P2Y₂ receptor subtype in purinergic Ca²⁺ signalling in the blood-brain barrier endothelial cell line hCMEC/D3 is proposed.