Objective: Osmotic stress induces a reorganization of the actin cytoskeleton that is important for the ensuing stress response. However, the mechanisms are incompletely understood, and the effect of osmotic stress on microtubules (MTs) essentially unstudied. End- binding proteins 1 and -3 (EB1 and EB3) are MT plus-end tracking proteins (+TIPs) that link MTs to multiple cellular structures and organelles, including the cell cortex. The aim of this study was to determine: (i) the effect of osmotic stress on MT organization, and (ii) the potential roles of EB1 and EB3 in osmotically induced cytoskeletal reorganization. Methods: Immunofluorescence microscopy, siRNA knockdown of EB1 or EB3, and quantitative F-actin assays were employed to evaluate effects of 50% osmotic shrinkage or swelling in interphase human retinal pigment epithelial (RPE) cells. Results: The number of visible MTs in wild type RPE cells was markedly reduced after 1 and 15 min of hypertonic shrinkage. Conversely, MT number was robustly increased after 1 and 15 min of hypotonic swelling. Shrinkage was associated with dissociation of EB1 and EB3 from MTs. Conversely, swelling elicited a marked increase in formation of EB1 and EB3 "comets", consistent with MT polymerization. Knockdown of either EB1 or EB3 had no effect on MT organization under iso- and hypertonic conditions, but lead to disorganization of the MT network after hypotonic swelling. Finally, preliminary data indicate that the rapid shrinkage-induced increase in F-actin content is abolished by EB1 or EB3 knockdown. Conclusion: In RPE cells, osmotic shrinkage elicits rapid MT depolymerisation and loss of EB1 and EB3 from MT tips. Conversely, osmotic swelling elicits rapid MT polymerization and EB1 and EB3 MT tip association. Swelling-induced MT polymerization and shrinkage-induced actin polymerization both appear to be dependent on EB1 and EB3. Thus, +TIPs may be novel players linking osmotic effects on MTs and the actin cytoskeleton.