12(S)-HETE, a lipoxygenase metabolite of arachidonic acid, has been demonstrated to induce a reversible retraction of vascular endothelial cells (EC). 12(S)-HETE-induced microvascular EC retraction was blocked by a selective protein kinase C inhibitor, calphostin C, but not by the protein kinase A inhibitor, H8. EC exposed to 12(S)-HETE demonstrated a gradual dissolution of actin microfilaments and a decrease of vinculin-containing focal adhesions. The intermediate filaments, vimentin, also underwent extensive reorganization (i.e., filament bundling and enrichment to the cell filapodia) following 12(S)-HETE treatment. In vivo phosphorylation studies revealed that 12(S)-HETE induced a hyperphosphorylation of several major cytoskeletal proteins including myosin light chain, actin, and vimentin. The increased phosphorylation of these cytoskeletal proteins following 12(S)-HETE stimulation was abolished by calphostin C but not by H8. Confluent EC express alpha v beta 3 in focal adhesions at both the cell body and the cell-cell borders. 12(S)-HETE induced a sequential rearrangement of the alpha v beta 3-containing focal adhesions, resulting in a general decrease in alpha v beta 3 integrin receptors, especially in those retracted EC. 12(S)-HETE-induced rearrangement of alpha v beta 3 was inhibited by calphostin C but not by H8. In contrast to alpha v beta 3, confluent EC enrich alpha 5 beta 1 integrin receptors primarily at the cell-cell borders, colocalizing with extracellular fibronectin and cell cortical microfilaments. 12(S)-HETE treatment also disrupted the cell-border distribution pattern of alpha 5 beta 1 as EC retracted, but no distinct alterations (such as time-related redistribution and quantitative differences) in alpha 5 beta 1 were observed.