Cell division, cell motility and the formation and maintenance of specialized structures in differentiated cells depend directly on the regulated dynamics of the actin cytoskeleton. To understand the mechanisms of these basic cellular processes, the signalling pathways that link external signals to the regulation of the actin cytoskeleton need to be characterized. Here we identify a pathway for the regulation of cofilin, a ubiquitous actin-binding protein that is essential for effective depolymerization of actin filaments. LIM-kinase 1, also known as KIZ, is a protein kinase with two amino-terminal LIM motifs that induces stabilization of F-actin structures in transfected cells. Dominant-negative LIM-kinasel inhibits the accumulation of the F-actin. Phosphorylation experiments in vivo and in vitro provide evidence that cofilin is a physiological substrate of LIM-kinase 1. Phosphorylation by LIM-kinase 1 inactivates cofilin, leading to accumulation of actin filaments. Constitutively active Rac augmented cofilin phosphorylation and LIM-kinase 1 autophosphorylation whereas phorbol ester inhibited these processes. Our results define a mechanism for the regulation of cofilin and hence of actin dynamics in vivo. By modulating the stability of actin cytoskeletal structures, this pathway should play a central role in regulating cell motility and morphogenesis.