Endothelial cells control water and solute flux between blood and tissue. Paracellular permeability can be regulated by via actomyosin contraction/relaxation. Elevated tension leads to paracellular gaps and a loss of barrier function. Extracellular ATP is a multifaceted signalling molecule implicated in the cellular mechano-response and in the regulation of vascular tone or permeability, the signalling mechanism of which is not yet clear. Fluid permeability usually is quantified via passage of macromolecules across cell layers - a procedure applicable only to cells cultured on permeable filter supports, which hamper optical microscopy. Moreover, the size of paracellular gaps in a functionally active culture is below the optical resolution limit. Therefore, we applied atomic force microscopy (AFM) as a high resolution tool to analyze 3D-surfaces of endothelial cells under physiological buffer conditions.We used primary cultures of endothelial cells isolated from human umbilical veins (HUVEC). Paracellular gaps could be quantitated to cover 0.57% of total area in control cells, samples treated with ionomycin (IM) (1mM) showed an increased gap area of 1.87%, while ATP (10mM) decreased the gap area to 0.31%. Moreover, contractile status was monitored in living cells using AFM-nanoindentation. Upon addition of IM, the cells stiffened transiently by 190%, while ATP led to cell softening of 27% as quantitated by the Young's modulus (YM). The contractile status was also reflected by the degree of phosphorylation of the myosin light-chain as confirmed by western blotting. The paracellular electric resistance across the layer was measured using impedance spectroscopy (ECIS). Analogously to the contractile status, the resistance was transiently decreased through IM by 34%, while ATP led to an increase of 24% above control level. In summary, paracellular permeability properties of cell layers can be assessed via AFM nanomechanical analysis demonstrating that ATP improves barrier function by an actomyosin-relaxation mechanism.