Mast cells undergo cytoskeletal restructuring to allow secretory granules passage through the cortical actomyosin barrier to fuse with the plasma membrane and release inflammatory mediators. Protein phosphorylation is believed to regulate these rearrangements. Although some of the protein kinases implicated in this phosphorylation are known, the relevant protein phosphatases are not. At the peak rate of antigen-induced granule mediator release (2.5 min), protein phosphatases PP1 and PP2A, along with actin and myosin II, are transiently relocated to ruffles on the apical surface and a band at the peripheral edge of the cell. This leaves an area between the nucleus and the peripheral edge significantly depleted (3-5-fold) in these proteins. Phorbol 12-myristate 13-acetate (PMA) plus A23187 induces the same changes, at a time coincident with its slower rate of secretion. Coimmunoprecipitation experiments demonstrated a significantly increased association of myosin with PP1 and PP2A at the time of peak mediator release, with levels of association decreasing by 5 min. Jasplakinolide, an inhibitor of actin assembly, inhibits secretion and the cytoskeletal rearrangements. Surprisingly, jasplakinolide also affects myosin, inducing the formation of short rods throughout the cytoplasm. Inhibition of PP2A inhibited secretion, the cytoskeletal rearrangements, and led to increased phosphorylation of the myosin heavy and light chains at protein kinase C-specific sites. These findings indicate that a dynamic actomyosin cytoskeleton, partially regulated by both PP1 and PP2A, is required for mast cell secretion.