In most mammalian cells, regulatory volume decrease (RVD) is mediated by swelling-activated Cl − and K + channels. Previous studies in the human neuroblastoma cell line CHP-100 have demonstrated that exposure to hypoosmotic solutions activates Cl − channels which are sensitive to Ca 2+. Whether a Ca 2+-dependent K + conductance is activated after cell swelling was investigated in the present studies. Reducing the extracellular osmolarity from 290 to 190 mOsm/kg H 2O rapidly activated 86Rb effluxes. Hypoosmotic stress also increased cytosolic Ca 2+ in fura-2 loaded cells. Pretreatment with 2.5 mM EGTA and nominally Ca 2+ free extracellular solution significantly decreased the hypoosmotically induced rise in cytosolic Ca 2+ and the swelling-activated 86Rb efflux. In cell-attached patch–clamp studies, decreasing the extracellular osmolarity activated a K + conductance that was blocked by Ba 2+. In addition, the swelling-activated K + channels were significantly inhibited in the presence of nominally free extracellular Ca 2+ and 2.5 mM EGTA. These results suggest that in response to hypoosmotic stress, a Ca 2+-dependent K + conductance is activated in the human neuroblastoma cell line CHP-100.