We have shown before that histamine induced endothelial calcium (Ca²⁺_i) waves are generated by a comparably low number of responder cells and transmitted via gap junctions. Since nitric oxide (NO) reduces gap junctional communication in HeLa cells expressing connexin 37 (Cx37) only we investigated, whether this effect could also be observed in HUVECs expressing a multitude of connexins (Cx37, and additionally, Cx40 and Cx43). Moreover, we studied the propagation of calcium waves between cultured endothelial and smooth muscle cells (both human umbilical vein derived cells) to account for "myoendothelial coupling" thought to be mediated mainly by Cx37. The effect on calcium wave transmission was taken as a readout for gap junctional coupling. Endogenous NO formation was suppressed by LNA to account for unknown differences in local formation and replaced exogenously by the NO donor SNAP (1mM). Calcium waves (detected using Fura2) were induced by mechanical stimulation of single cells in endothelial monolayers. In some experiments, the expression of Cx43 was reduced by siRNA treatment against Cx43, as assessed by Western blotting. Mechanical stimulation of endothelial cells increased their intracellular calcium levels and calcium rapidly spread to > 75% of the adjacent cells (AC, N=50, 8 wells, 3 cultures) and 50% of secondary adjacent cells (SAC, N=100, 8 wells, 3 cultures). SNAP significantly reduced the propagation velocity and prolonged the time interval till onset of calcium signals in SAC (N=80, 8 wells, 3 cultures). In cells pre-treated with siRNACx43 intracellular calcium increased only in 51% of AC (control siRNA: 83%, N=12, 4 wells, 2 cultures). Between endothelial and smooth muscle cells, the amplitude of the Ca²⁺_i increase and the propagation velocity decreased significantly by a factor 2 (SMC -> HUVEC) when treated with the NO donor SNAP (N=29, 4wells, 2 cultures). Our results are consistent with the hypothesis that NO inhibits Cx37-dependent GJC and calcium wave propagation in endothelial cells. This effect is masked by compensatory propagation through gap junctions formed by Cx43. In myoendothelial gap junctions, the inhibitory NO effect is already visible when all three endothelial connexins are present. Therefore, the NO effect on calcium wave propagation may be functionally most important in myoendothelial junctions.