The effect of bacterial flora on cytokine production from resident peritoneal macrophages was investigated in the mouse. The production of IL-1, IL-6 and TNF-alpha was determined in germ-free, and "conventionalized" mice, as well as in monoxenic mice implanted with either the Gram-negative bacterium E. coli, or the Gram-positive organism Bifidobacterium bifidum. Macrophages from the "conventionalized" mice produced significantly more IL-1 and IL-6 in vitro than those of the germ-free mice. IL-1 and IL-6 production from germ-free mice implanted with E. coli was comparable to that from "conventionalized" mice. However, implantation with Bifidobacterium bifidum did not increase production of these two cytokines above levels observed for macrophages from the germ-free mice. A little TNF-alpha was produced by only the macrophages from the "conventionalized" and monoxenic mice implanted with E. coli. Soon after implantation, the bacterial flora stimulated cytokine production by mouse peritoneal macrophages and our results suggest that Gram negative bacteria are the most efficient stimulus for this production.