Background: The immunomodulating potential residing in cow's milk proteins is currently receiving increasing attention because of growing interest in functional foods and the complex problem of cow's milk allergy. One of the major cow's milk allergens, whey protein β-lactoglobulin, has previously been shown to mediate cellular activation in both human and murine immune cells. Objective: We examined the response to different β-lactoglobulin preparations in naive immune cells. Methods: Splenocytes and cells from mesenteric lymph nodes derived from BALB/c mice bred and maintained on a milk-free diet were cultured in vitro with different β-lactoglobulin preparations. Cell proliferation, cytokine production, and increases in intracellular glutathione were used as cellular activation markers. Moreover, the effect of β-lactoglobulin on cytokine production in murine bone-marrow-derived dendritic cells was examined. Results: We observed that some commercial β-lactoglobulin preparations induced pronounced proliferation of both spleen cells and cells from mesenteric lymph nodes; production of TNF-α, IL-6, IL-1β, and IL-10; and an increased level of intracellular glutathione in spleen cell cultures. Furthermore, TNF-α, IL-6, IL-1β, and IL-10 production was induced in murine bone-marrow-derived dendritic cells. Purification of β-lactoglobulin from raw milk using nondenaturating conditions, however, revealed that the β-lactoglobulin per se did not possess the immunomodulatory activity. Eventually, the immunostimulatory effect was found to be caused by endotoxin contamination. Conclusion: These results identify endotoxin as the main immunostimulatory component present in some commercial β-lactoglobulin preparations. Moreover, the present study makes it evident that immunomodulatory effects attributed to β-lactoglobulin need to be reassessed.