To investigate the role of PTEN in regulation of cortical motile activity, especially in myosin II localization, eGFP-PTEN and mRFP-myosin II were simultaneously expressed in Dictyostelium cells. PTEN and myosin II co-localized at the posterior of migrating cells and furrow region of dividing cells. In suspension culture, PTEN knockout (pten(-)) cells became multinucleated, and myosin II significantly decreased in amount at the furrow. During pseudopod retraction and cell aspiration by microcapillary, PTEN accumulated at the tips of pseudopods and aspirated lobes prior to the accumulation of myosin II. In pten(-) cells, only a small amount of myosin II accumulated at the retracting pseudopods and aspirated cell lobes. PTEN accumulated at the retracting pseudopods and aspirated lobes even in myosin II null cells and latrunculin B-treated cells though in reduced amounts, indicating that PTEN accumulates partially depending on myosin II and cortical actin. Accumulation of PTEN prior to myosin II suggests that PTEN is an upstream component in signaling pathway to localize myosin II, possibly with mechanosensing signaling loop where actomyosin-driven contraction further augments accumulation of PTEN and myosin II by a positive feedback mechanism.