Signal transduction processes in T-cells and other cell types alter the phosphorylation state of cofilin, an actin-binding phosphoprotein. Whether reversible phosphorylation is responsible for the regulation of the functional activities of cofilin is not clear at present. Here we have identified the phosphoacceptor site of cofilin and analyzed the role of cofilin phosphorylation with respect to its subcellular localization. Site-directed mutagenesis studies show that phosphorylation occurs exclusively on Ser-3. Expression of non-phosphorylatable mutant cofilin proteins in NIH3T3 cells and determination of their subcellular localization by confocal laser scanning microscopy reveal that non-phosphorylated cofilin accumulates within nuclei. This analysis shows that the subcellular localization of cofilin depends on the phosphorylation state of Ser-3.