Migration of epithelial cell sheets, a process involving F-actin restructuring through Rho family GTPases, is both physiologically and pathophysiologically important. Our objective was to clarify the mechanisms whereby the downstream RhoA effector ROCK influences coordinated epithelial cell motility. Although cells exposed to a pharmacologic ROCK inhibitor (Y-27632) exhibited increased spreading in wound closure assays, they failed to migrate in a cohesive manner. Two main phenomena were implicated - the formation of aberrant protrusions at the migrating front, and the basal accumulation of F-actin aggregates. Aggregates reflected increased membrane affiliation and detergent-insolubility of the actin-binding protein ezrin, and enhanced co-association of ezrin with the membrane protein CD44. While F-actin aggregation following ROCK inhibition was recapitulated by inhibiting myosin light chain phosphorylation with the MLCK inhibitor ML-7, the latter did not influence protrusiveness and in fact significantly decreased cell migration. Our results suggest that excessive protrusiveness downstream of ROCK inhibition reflect an influence of ROCK on F-actin stability via LIM-kinase 1 (LIM-K1), which phosphorylates and inactivates cofilin. Y-27632 reduced levels of both active LIM-K1 and inactive cofilin (phospho-forms); and expression of a dominant-negative LIM-K1 mutant stimulated leading edge protrusiveness. Furthermore, Y-27632-induced protrusions were partially reversed by over-expressing LIM-K1 to restore cofilin phosphorylation. In summary, our results provide new evidence suggesting that adhesive and protrusive events involved in organised epithelial motility downstream of ROCK are separately coordinated through the phosphorylation of (respectively) MLC and cofilin.