Protein phosphorylation/dephosphorylation and cytoskeletal reorganization regulate the Na⁺,K⁺,2Cl^- cotransporter (NKCC1) during osmotic shrinkage, however, the mechanisms involved are unclear. Here we show that in cytoplasts (plasma membrane vesicles detached from Ehrlich Ascites Tumor cells (EATC) by cytochalasin treatment) NKCC1 activity evaluated as bumetanide-sensitive ⁸⁶Rb-influx, was increased compared to the basal level in intact cells, yet could not be further increased by osmotic shrinkage. Accordingly, cytoplasts exhibited no regulatory volume increase after shrinkage. In cytoplasts, cortical F-actin organization was disrupted, and myosin II, which in shrunken EATC translocates to the cortical region, was absent. Moreover, NKCC1 activity was essentially insensitive to the MLCK inhibitor ML-7, a potent blocker of shrinkage-induced NKCC1 activity in intact EATC. Cytoplast NKCC1 activity was potentiated by the Ser/Thr protein phosphatase inhibitor calyculin A, partially inhibited by the protein kinase A inhibitor H89, and blocked by the broad protein kinase inhibitor staurosporine. Cytoplasts exhibited increased protein levels of NKCC1, Ste20-related proline-and-alanine-rich kinase (SPAK) and oxidative stress response-kinase (OSR1), yet lacked the shrinkage-induced plasma membrane translocation of SPAK observed in intact cells. The basal phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) was increased in cytoplasts compared to intact cells, yet in contrast to the substantial activation in shrunken intact cells, p38 MAPK could not be further activated by shrinkage of the cytoplasts. Taken together these findings indicate that shrinkage-activation of NKCC1 in Ehrlich Ascites Tumor cells is dependent on the cortical F-actin network, myosin II, and MLCK.