Lactococcus lactis subsp. lactis G50 has immunomodulatory activity and is a candidate for use as a probiotic strain. We investigated the factors that affect the immunomodulatory activity of this strain. The macrophage-like cell line J774.1A was exposed to live or dead cells of strain G50 grown in different media, and the interleukin (IL) 12 produced by the cell line was then measured. Live cells grown in M17 supplemented with glucose (GM17 cells) induced IL-12 production by J774.1 cells significantly more than did cells grown in deMan Rogosa Sharpe (MRS) broth (MRS cells; P < 0.05). In the case of dead cells, the opposite results were obtained in these two samples. The sugar content of GM17 cells was significantly higher than that of MRS cells (P < 0.01). The fatty acid compositions of GM17 cells and MRS cells differed. Lysis of GM17 cells by lysozyme, which degrades the cell wall, was greater than in MRS cells. The cell wall fraction prepared from GM17 cells induced significantly more IL-12 production than did the fraction from MRS cells (P < 0.05). These results indicated that alterations in cellular components or in the structure of the cell surface by the growth media affected the immunomodulatory activity of strain G50. Attention should be paid to the selection of growth medium in testing for the immunomodulatory activity of lactic acid bacteria.