Hypoosmotic shock treatment increased cytosolic Ca2+ ion concentration ([ca2+],,,) in tobacco (Nicotiana fabacum) suspensionculture cells. [CaZ+],,, measurements were made by genetically transforming these cells to express apoaequorin and by reconstituting the Caz+-dependent photoprotein, aequorin, i n the cytosol by incubation with chemically synthesized coelenterazine. Measurement of Ca2+-dependent luminescence output thus allowed the direct monitoring of [Ca2+lcy, changes. When cells were added to a hypoosmotic medium, a biphasic increase in ICa2+],,, was observed; an immediate small elevation (phase 1) was observed first, followed by a rapid, large elevation (phase 2). Phase 1 [Ca2+],,, was stimulated by the V-type ATPase inhibitor bafilomycin A,. Phase 2 was inhibited by the protein kinase inhibitor K-252a and required the continued presence of the hypoosmotic stimulus to maintain it.Although Ca2+ in the medium was needed to produce phase 2, it was not needed to render the cells competent to the hypoosmotic stimulus. If cells were subject to hypoosmotic shock in CaZ+-depleted medium, increases in luminescence could be induced up to 20 min after the shock by adding Ca2+ to the medium. These data suggest that hypoosmotic shock-induced [Ca2+l,,, elevation results from the activity of a Ca2+ channel i n the plasma membrane or associated hypoosmotic sensing components that require Ca2+-independent phosphorylation and a continued stimulus to maintain full activity.The maintenance and regulation of turgor pressure is essential for many aspects of plant growth, development, and movements. Although turgor pressure can be regulated via the synthesis and degradation of osmotic protectants, rapid changes usually occur by modifications of the flux of inorganic ions across the plasma membrane (Schroeder and Hedrich, 1989).A great deal of research effort has been directed at investigating the responses of higher plants to hyperosmotic signals (Greenway and Munns, 1980), because hyperosmotic stress during salination or drought, for example, is