ADF/cofilin is a phosphorylation-regulated protein essential for actin filament dynamics in cells. Here, we cloned two cDNAs encoding Xenopus ADF/cofilin (XAC)-specific phosphatase, slingshot (XSSH), one of which contains an extra 15 nucleotides in a coding sequence of the other, possibly generated by alternative splicing. Whole mount in situ hybridization showed XSSH transcripts in the blastopore lip and sensorial ectoderm at stage 11, and subsequently localized to developing brain, branchial arches, developing retina, otic vesicle, cement gland, and spinal chord in neurula to tailbud embryos. Immunostaining of animal-vegetal sections of gastrula embryos demonstrated that both XAC and XSSH proteins are predominant in ectodermal and involuting mesodermal cells. Microinjection of either a wild type (thus induces overexpression) or a phosphatase-defective mutant (functions as dominantly negative form) resulted in defects in gastrulation, and often generated the spina bifida phenotype with reduced head structures. Interestingly, the ratio of phosphorylated XAC to dephosphorylated XAC markedly increased from the early gastrula stage (stage 10.5), although the amount of XSSH protein markedly increased from this stage. These results suggest that gastrulation movement requires ADF/cofilin activity through dynamic regulation of its phosphorylation state.