A substance, active as a B-cell mitogen, was isolated from the slime products produced by Lactococcus lactis ssp. cremoris KVS20. The mitogenic substance was prepared by anion-exchange chromatography and gel filtration chromatography and then purified by proteinase digestion and HPLC. Chemical analysis determined that the mitogenic substance was a phosphopolysaccharide and consisted of rhamnose, glucose, galactose, and phosphorus. The activity of the mitogenic substance was higher than that of the slime products. The optimal concentration for the activity was approximately 120 micrograms/ml. The mitogenic substance also had substantial mitogenic activity to spleen cells from C3H/HeJ mice, which are resistant to lipopolysaccharide. The findings indicated that a B-cell mitogen different from lipopolysaccharide is produced from L. lactis ssp. cremoris KVS20.