Osmolytes accumulated in the osmotically stressed Yersinia enterocolitica ATCC 9610 were investigated using natural abundance ^(13)C NMR spectroscopy. Glycine betaine, one of the more common and most effective osmolytes found in nature, was the dominant osmolyte in osmotically stressed Y. enterocolitica cells. Glycine betaine concentration was 41.8 times higher (801.9 nmol/㎎ protein) in stressed cells than in unstressed cells (19.2 nmol/㎎ protein). Proline was the minor osmolyte, and its concentration was 284.8 nmol/㎎ protein. The effects of glycine betaine and related osmolytes on growth rate of osmotically stressed Y. enterocolitica were investigated to identify their ability as osmolytes for Y. enterocolitica. When glycine betaine and proline were added in MMA medium containing 2.5% NaCl, the growth rate with glycine betaine (1 mM) was 3.6 times higher than in control (no addition of osmolyte), and that with proline was 1.3 times higher. Dimethylglycine (5 mM) also increased the growth rate 3.1 folds. On the other hand, monomethylglycine had no effect on growth of osmotically stressed and unstressed Y. enterncolitica. When carnitine was added in MMA medium containing 2.5% NaCl, carnitine (5 mM) increased the growth rate 2.4 folds, but choline had no effect on growth of osmotically stressed Y. enterocolitica. The above result indicate that glycine betaine is the dominant osmolyte in osmotically stressed Y. enterocolitica, and proline, dimethylglycine and carnitine also act as minor osmolytes.