AbstractBackground:  Cofilin, a key regulator of actin filament dynamics, is inactivated by phosphorylation at Ser‐3 by LIM‐kinases and is reactivated by dephosphorylation by a family of protein phosphatases, termed Slingshot (SSH).Results:  We have identified two novel isoforms of SSHs, termed SSH‐2L and SSH‐3L and characterized them in comparison with SSH‐1L that was previously reported. SSH‐1L and SSH‐2L, but not SSH‐3L, tightly bound to and co‐localized with actin filaments. When expressed in cultured cells, SSH‐1L, SSH‐2L and SSH‐3L decreased the level of Ser‐3‐phosphorylated cofilin (P‐cofilin) in cells and suppressed LIM‐kinase‐induced actin reorganization, although SSH‐3L was less effective than SSH‐1L and SSH‐2L. In cell‐free assays, SSH‐1L and SSH‐2L efficiently dephosphorylated P‐cofilin, whereas SSH‐3L did do so only weakly. Using deleted mutants of SSH‐1L and SSH‐2L, we found that the N‐terminal and C‐terminal extracatalytic regions are critical for cofilin‐phosphatase and F‐actin‐binding activities, respectively. In situ hybridization analyses revealed characteristic patterns of expression of each of the mouse Ssh genes in both neuronal and non‐neuronal tissues; in particular, expression of Ssh‐3 in epithelial tissues is evident.Conclusion:  SSH‐1L, SSH‐2L and SSH‐3L have the potential to dephosphorylate P‐cofilin, but subcellular distribution, F‐actin‐binding activity, specific phosphatase activity and expression patterns significantly differ, which suggests that they have related but distinct functions in various cellular and developmental events.