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Prebiotics and probiotics could represent an effective alternative to the use of synthetic antibiotics in nutrition. The mechanisms by which prebiotics affect the immune system have not yet been investigated in detail. Most effects have been attributed to increases in the innate and acquired immune responses. This study was conducted to elucidate the long-term effects of orally administered lactulose on the immune response in the intestinal tract of probiotic-fed calves. Preruminant calves were randomized to 3 feeding groups: milk replacer containing 1) no lactulose, 2) 1% lactulose, or 3) 3% lactulose. All 3 milk replacers contained 10(9) cfu Enterococcus faecium/kg. Messenger RNA expression of different cell activation markers, pro- and antiinflammatory cytokines, and IgA Fc receptor was investigated in the ileum, mesenterial lymph node, spleen, and white blood cells. A significantly greater number of blood lymphocytes were detected in the 3% lactulose group (P = 0.02) than in the control group. The expression results in male calves indicated that the transcription of IgA Fc receptor in the ileal mucosa of the 1% lactulose treatment group increased significantly (P = 0.04) and also tended to increase in the 3% lactulose group (P = 0.07). Furthermore, decreases in IL-10 and interferon-gamma mRNA expression were observed in the ileum (P = 0.04). The CD4-presenting lymphocytes were decreased significantly in the ileum (P = 0.04) and mesenteric lymph node (P = 0.01), whereas CD8-presenting lymphocytes were increased in the blood (P = 0.03) of females. Other proinflammatory cytokines (IL-1beta, IL-8, and tumor necrosis factor-alpha) and antiinflammatory cytokines (transforming growth factor-beta1) did not show significant differences in mRNA expression among treatment groups. The results indicate that additional lactulose feeding had an immunomodulatory effect on the composition of T-cell subsets in different immune compartments and had minor effects on pro- and antiinflammatory cytokine mRNA expression.