Intestinal Bifidobacterium species are thought to be beneficial in animal and human intestines. We studied the mechanisms of Bifidobacteria in antitumor activity using a cell wall preparation (WPG) of B. infantis (Cancer Res., 45, 1300, (1985)). WPG enhanced the in vitro antitumor activities of mouse peritoneal exudate cells elicited with proteose-peptone (P-PEC) and thioglycollate broth (TG-PEC), determined by cytostatic ([³H] thymidine uptake inhibition) and cytolytic ([³H] uridine release) assays. Tumor necrosis factor-α (TNF-α) and reactive nitrogen intermediates (RNI) play a role in such augmented cytotoxicity, because anti-TNF-α antibody almost completely blocked the increased cytolytic activity of P-PEC in the presence of WPG. Moreover, WPG induced RNI in the supernatant of TG-PEC in a dose-dependent manner. The mRNA expression of several cytokines (IL-1β, IL-6, IL-10, IFN-α and TNF-α) was induced in BALB/c mouse peritoneal cells 3 h after an intraperitoneal injection of WPG (3 h WPG-PEC). However, this expression disappeared from 24 h WPG-PEC, except for that of IFN-α. IFN-γ was not induced. Kinetic studies of the tumor neutralizing activities of the WPG-PECs by means of the in vivo Winn assay revealed that the activity emerged at 1.5 h, became maximal at 3 h and disappeared at 24 h. These results indicated that Bifidobacterial WPG is a Biological Response Modifier (BRM) with characteristics similar to those of other bacterial BRMs.