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 Ca2+. Whether a Ca2+-dependent K+ conductance is activated after cell swelling was investigated in the present studies. Reducing the extracellular osmolarity from 290 to 190 mOsm/kg H2O rapidly activated 86Rb effluxes. Hypoosmotic stress also increased cytosolic Ca2+ in fura-2 loaded cells. Pretreatment with 2.5 mM EGTA and nominally Ca2+ free extracellular solution significantly decreased the hypoosmotically induced rise in cytosolic Ca2+ and the swelling-activated 86Rb efflux. In cell-attached patch–clamp studies, decreasing the extracellular osmolarity activated a K+ conductance that was blocked by Ba2+. In addition, the swelling-activated K+ channels were significantly inhibited in the presence of nominally free extracellular Ca2+ and 2.5 mM EGTA. These results suggest that in response to hypoosmotic stress, a Ca2+-dependent K+ conductance is activated in the human neuroblastoma cell line CHP-100.