The quaternary ammonium compounds accumulated in saline conditions by five salt-tolerant species of Limonium (Plumbaginaceae) were analyzed by fast atom bombardment mass spectrometry. Three species accumulated j-alanine betaine and choline-O-sulfate; the others accumulated glycine betaine and choline-C-sulfate. Three lines of evidence indicated that f,-alanine betaine and choline-0-sulfate replace glycine betaine as osmoregulatory solutes. First, tests with bacteria showed that 0-alanine betaine and choline-0-sulfate have osmoprotective properties comparable to glycine betaine. Second, when ,B-alanine betaine and glycine betaine accumulators were salinized, the levels of their respective betaines, plus that of choline-0-sulfate, were closely correlated with leaf solute potential. Third, substitution of sulfate for chloride salinity caused an increase in the level of choline-C-sulfate and a matching decrease in glycine betaine level. Experiments with '4C-labeled precursors established that B-alanine betaine accumulators did not synthesize glycine betaine and vice versa. These experiments also showed that 6-alanine betaine synthesis occurs in roots as well as leaves of fialanine betaine accumulators and that choline-C-sulfate and glycine betaine share choline as a precursor. Unlike glycine betaine, j-alanine betaine synthesis cannot interfere with conjugation of sulfate to choline by competing for choline and does not require oxygen. These features of #l-alanine betaine may be advantageous in sulfate-rich salt marsh environments.Considerable evidence indicates that the QAC2 glycine betaine accumulates in the cytoplasm and chloroplasts of species of the family Chenopodiaceae during osmotic stress and acts as a nontoxic or compatible osmolyte (for reviews, see refs. 15 and 28). Glycine betaine is likely to play the same role in the many other angiosperm families in which it accumulates in response to stress (27,30), although in these cases the '