Soy protein hydrolysates with lower molecular weight were enzymatically prepared by several commercially available proteases (Alcalase 2.4L, Flavourzyme, Trypsin, Papain, Protease A and Peptidase R) with protein recovery varied from 42.59% to 79.87%. Relative content of positively charged peptides was determined on SP Sephadex C-25 using gradient sodium chloride solution as eluents. Immunomodulating properties were evaluated by measuring their effect on in vitro proliferation of murine spleen lymphocytes and phagocytic activity of peritoneal macrophages. The results showed that soy protein hydrolysates (SPHs) prepared with Alcalase and insoluble soy protein (InSP), preferable to other enzymes and soy proteins, have the highest immunomodulating activity and the optimum conditions were determined as follows: E/S=2% (Alcalase), 60 degrees C, pH 8.0, InSP concentration 6% and 225min. Positive correlations were obtained between the immunomodulating activity and content of positively charged peptides. The results suggested that lower molecular weight and positively charged peptides released from soy protein were effective in stimulating immunomodulating activity, thus provided insights into the preparation of potent immunomodulating products.