Activation of PAR-1 in bovine corneal endothelial cells (BCEC) by thrombin leads to the formation of stress fibers and intercellular gaps. This results in loss of barrier integrity and inhibition of intercellular communication (IC). Previous studies in our laboratory demonstrated that thrombin inhibits intercellular propagation of calcium waves and blocks uptake of Lucifer Yellow (LY) via hemichannels that are involved in paracrine IC. Our studies provided evidence that the effect of thrombin is mediated by an increase in MLC phosphorylation. Since the main effect of MLC phosphorylation is enhanced contractility, we investigated whether inhibition of myosin II ATPase activity by (-)-blebbistatin affects the thrombin-induced reduction of IC. IC was investigated by studying calcium wave propagation and LY dye uptake in confluent monolayers of primary cultures of BCEC. Calcium waves, elicited by applying a mechanical stimulus to a single cell, were visualized by confocal microscopy using Fluo-4AM. The spread of the wave was quantified by measuring the active area. LY dye uptake in calcium free conditions (2mM EGTA, 5 min) was also visualized by confocal fluorescence microscopy. In six independent experiments (with eight calcium waves per condition), the active areas of cells treated with thrombin (2 U/ml, 5 min) was significantly lower in the absence (17.7  1.6 × 10³ mm²) than in the presence (37.6  2.5 × 10³ mm²) of (-)-blebbistatin (5 mM, 20 min) (n = 48). No significant differences in active area were found between cells in control condition, in the presence of thrombin plus (-)-blebbistatin, and in the presence of (-)-blebbistatin alone. Cells treated with thrombin after pretreatment with (-)-blebbistatin clearly show LY dye uptake. (-)-Blebbistatin did not significantly affect the enhancement of MLC phosphorylation by thrombin. In conclusion, pretreatment of BCEC with (-)-blebbistatin prevented the inhibitory effect of thrombin on calcium wave propagation and LY uptake. These findings provide evidence that myosin II mediated contractile forces play an important role in the inhibitory mechanism of thrombin on IC.