The aim of this work was to study the physiological response of Lactobacillus plantarum subjected to osmotic stress in the presence of three structurally related compatible solutes, Either betaine, choline or acetylcholine was accumulated by osmotically stressed cells when provided in the chemically defined medium. Choline and acetylcholine were accumulated to maximum concentrations of 139 and 222 mu mol g (dry weight) of cells(-1) respectively and were not converted to betaine. Addition of 0.5 mM choline or 0.5 mM acetylcholine to the medium increased the growth rates of cells in media with various amounts of added sodium chloride, Both choline and acetylcholine are positively charged compounds : therefore, it was presumed that charged intracellular solutes could counterbalance the excess of positive charge, Intracellular inorganic ion levels (K+, SO42-, PO43- and Cl-) of cells cultured under conditions of osmotic stress remained similar in the presence of either betaine, choline or acetylcholine, However, cells cultured ill the presence of choline or acetylcholine accumulated an additional quantity of approximately 125 or 200 mu mol.glutamate (dry weight) cells(-1) respectively, as compared to cells grown in the presence of betaine. Hence glutamate appears to be the counterion for choline and acetylcholine. This is the first study demonstrating accumulation of choline and acetylcholine in lactic acid bacteria subjected to osmotic stress.