Glycinebetaine is an osmoprotectant accumulated by barley (Hordeum vulgare) plants in response to high levels of NaCl, drought, and cold stress. Using barley seedlings in hydroponic culture, we characterized additional inducers of glycinebetaine accumulation. These included other inorganic salts (KCl, MgCl(2), LiCl, and Na(2)SO(4)), oxidants (H(2)O(2) and cumene hydroperoxide), and organic compounds (abscisic acid, polymixin B, n-butanol, salicylic acid, and aspirin). Stress symptoms brought on by high NaCl and other inducers, and not necessarily correlated with glycinebetaine accumulation, include wilting, loss of chlorophyll, and increase in thiobarbituric acid reacting substances. For NaCl, Ca(2+) ions at 10 to 20 mM decrease these stress symptoms without diminishing, or even increasing, glycinebetaine induction. Abscisic acid induces glycinebetaine accumulation without causing any of the stress symptoms. NaCl, KCl, and H(2)O(2) (but not other inducers) induce glycinebetaine at concentrations below those needed for the other stress symptoms. Mg(2+) at 10 to 20 mM induces both stress symptoms and glycinebetaine, but only at low (0.2 mM) Ca(2+). Although illumination is needed for optimal induction, a significant increase in the leaf glycinebetaine level is found in complete darkness, also.