Thrombin inhibits intercellular communication (IC) in bovine corneal endothelial cells (BCEC) via enhanced MLC phosphorylation. Adenosine can prevent this thrombin-induced inhibition of intercellular calcium wave propagation. Previous experiments also showed that the thrombin-effect can be abolished by the MLC-kinase inhibitor ML-7, the PKC inhibitor chelerythrine, and the Rho-kinase inhibitor Y-27632, thereby confirming the importance of MLC phosphorylation in the effect of thrombin on IC. In this study, we have investigated MLC phosphorylation in BCEC after thrombin and adenosine treatment to correlate it with our IC data. For this purpose, we have visualized mono- (Ser19) and diphosphorylation (Thr18/Ser19) by immunocytochemistry using confocal fluorescence microscopy (Zeiss LSM510). Confluent BCEC monolayers treated with thrombin and/or adenosine, were fixed and then stained with phospho-MLC2 specific antibodies (Cell Signaling Technology^TM, #3671, #3674). Ten representative high-resolution images were acquired for each condition by focusing on the cortical actin ring, stained by fluorescently labeled phalloidin. Then, the relative mean fluorescence intensity (MFI) per condition was calculated as a ratio (treated/control) for each experiment. In four independent experiments thrombin (2 U/ml, 5 min) treatment induced a significant increase in MFI of mono- (1.57  0.18, N = 4) and diphosphorylated (2.46  0.19, N = 4) MLC. In thrombin-treated cells, pre-incubation with adenosine (200 mM, 30 min) prevented the thrombin-induced increase in MFI of diphosphorylated (1.34  0.08, N = 4) MLC. While adenosine reduced the effect of thrombin on monophosphorylation, the effect was not significant. However, the monophosphorylation of MLC in the presence of adenosine plus thrombin was not significantly different from control condition. These findings are in line with previous studies by Western blotting. Confocal immunofluorescence microscopy showed a significant increase in mono- and diphosphorylation of MLC in thrombin-treated BCEC. This increase in phosphorylation can be significantly reduced by pre-incubating BCEC with adenosine.