The Galpha subunits of the G(12) family of heterotrimeric G proteins, defined by Galpha(12) and Galpha(13), are involved in many signaling pathways and diverse cellular functions. In an attempt to elucidate downstream effectors of Galpha(12) for cellular functions, we have performed a yeast two-hybrid screening of a rat brain cDNA library and revealed that Ser/Thr protein phosphatase type 5 (PP5) is a novel effector of Galpha(12) and Galpha(13). PP5 is a newly identified phosphatase and consists of a C-terminal catalytic domain and an N-terminal regulatory tetratricopeptide repeat (TPR) domain [2]. Arachidonic acid was recently shown to activate PP5 phosphatase activity by binding to its TPR domain, however the precise regulatory mechanism of PP5 phosphatase activity is not fully determined. In this study, we show that active forms of Galpha(12) and Galpha(13) specifically interact with PP5 through its TPR domain and activate its phosphatase activity about 2.5-fold. Active forms of Galpha(12) and Galpha(13) also enhance the arachidonic acid-stimulated PP5 phosphatase activity about 2.5-fold. Moreover, we demonstrate that the active form of Galpha(12) translocates PP5 to the cell periphery and colocalizes with PP5. These results propose a new signaling pathway of G(12) family G proteins.