The principal objective of this study was to compare the effects of whole and hydrolyzed cells (bifidobacteria) treated with gastrointestinal digestive enzymes on the activation of cloned macrophages. Seven different strains of Bifidobacterium obtained from swine, chickens, and rats, were digested with pepsin followed by pancreatin and the precipitate (insoluble fraction) and supernatant (soluble fraction) obtained via centrifugation. The RAW 264.7 murine macrophages were incubated with either whole cells, the precipitate, or supernatant at various concentrations. Pronounced increases in the levels of nitric oxide (NO), interleukin (IL)-1beta, IL-6, IL-12, and tumor necrosis factor (TNF)-alpha were observed in the whole cells and precipitates, but these effects were less profound in the supernatants. The precipitates also evidenced a slight, but significant, inductive activity for NO and all tested cytokines, with the exception of TNF-alpha in the macrophage model as compared with the whole cells. By way of contrast, TNF-alpha production when cultured with whole cells (100 ng/ml) resulted in marked increases as compared with what was observed with the precipitates. The results of this study indicated, for the first time, that digested Bifidobacterium sp. can induce the production of NO and several cytokines in RAW 264.7 murine macrophage cells. In the current study, it was demonstrated that Bifidobacterium strains treated with digestive enzymes, as compared with whole cells, are capable of stimulating the induction of macrophage mediators, which reflects that they may be able to modulate the gastrointestinal immune functions of the host.