Although the signaling pathways leading to hydrogen peroxide (H2O2)-induced endothelial monolayer permeability remain ambiguous, cytoskeletal proteins are known to be essential for maintaining endothelial integrity and regulating solute flux through the monolayer. We have recently demonstrated that thrombin-induced actin reorganization in bovine pulmonary artery endothelial cells (BPAEC) requires activation of both myosin light chain kinase (MLCK) and protein kinase C (PKC). Therefore, the present study was designed to investigate the effects of H2O2 on actin reorganization in BPAEC. H2O2 initiated sustained recruitment of actin to the cytoskeleton and transient myosin recruitment in a time- and concentration-dependent manner. The H2O2-induced actin recruitment was significantly inhibited by the calmodulin antagonists, W7 and TFP, but not by the MLCK inhibitor, KT5926, nor the PKC inhibitors, H7 and calphostin C. H2O2 also caused actin filament rearrangement in BPAEC with disruption of the dense peripheral bands and formation of stress fibers. These alterations occurred prior to actin translocation to the cytoskeleton and are prevented by inhibition of either MLCK or PKC. High concentrations of H2O2 transiently attenuated PKC activity but slightly increased the phosphorylation of the prominent PKC substrate and actin-binding protein, myristoylated alanine-rich C kinase substrate (MARCKS), by 5 min. However, MARCKS phosphorylation was reduced to below basal levels by 30 min. On the other hand, H2O2 induced a time- and dose-dependent phosphorylation of myosin light chains which was eliminated by both MLCK and PKC inhibitors. These data suggest that MLCK contributes to H2O2-induced myosin light chain phosphorylation and actin rearrangement and that PKC may play a permissive role. Neither of these enzymes appears to be involved in the H2O2-induced recruitment of actin to the cytoskeleton.