Thrombin inhibits gap junctional (GJIC) and paracrine communication (PIC) in bovine corneal endothelial cells (BCEC) via enhanced MLC phosphorrylation, suggesting involvement of the myosin II ATPase in this mechanism. In this study, we investigated involvement of myosin II ATPase activity in the inhibitory mechanism of thrombin on GJIC and PIC. Fluorescence recovery after photobleaching (FRAP) was used to investigate GJIC. ATP release through hemichannels, which was shown to mediate PIC in BCEC, was measured by the luciferin-luciferase bioluminescence assay. Calcium wave propagation assays in presence of connexin-mimetic peptides (namely Gap26 and Gap27) were used to separate PIC and GJIC. The reduction of FRAP by thrombin, which we previously demonstrated, was completely prevented upon preincubation with (-)-blebbistatin. Also the reduction of ATP release upon mechanical stimulation by thrombin was prevented by preincubation with (-)-blebbistatin. The reduction of active area of calcium wave propagation in the presence of Gap27, a connexin mimetic peptide that inhibits GJIC, was also prevented by (-)-blebbistatin. In the absence of thrombin, (-)-blebbistatin had no significant effect on either PIC or GJIC. The inactive enantiomer (+)-blebbistatin did not preclude the effect of thrombin on GJIC or PIC.To exclude confounding of our experiments by photoinactivation of blebbistatin, the effects of photoinactivated (-)-blebbistatin and (-)-nitroblebbistatin were tested. Photoinactivated (-)-blebbistatin had no effect on GJIC and PIC, while (-)-nitroblebbistatin exerted the same effect as (-)-blebbistatin. Therefore we can conclude that inhibition of myosin II ATPase activity prevents the effects of thrombin on GJIC and PIC. These findings provide evidence that myosin II mediated contractile forces play an important role in the inhibitory mechanism of paracrine and gap junctional communication.