Germfree F344 rats developed severe arthritis with 100% incidence after a single intradermal injection of either squalane containing 0.5 mg of heat-killed Mycobacterium bovis BCG or a water-in-oil emulsion containing 0.2 mg of peptidoglycan derived from Staphylococcus epidermidis. Conventional F344 rats developed less-severe arthritis with 20% incidence for heat-killed BCG and 0% incidence for peptidoglycan. Specific-pathogen-free rats showed an intermediate susceptibility between germfree and conventional rats. Interestingly, both unimmunized specific-pathogen-free and conventional rats, but not unimmunized germfree rats, showed weak delayed-type hypersensitivity reactions to peptidoglycans derived from either S. epidermidis or Lactobacillus plantarum, suggesting that a bacterial flora may furnish a stimulus for induction of cell-mediated immunity to ubiquitous bacterial peptidoglycans. It is thus possible that although a bacterial flora is not necessary for development of adjuvant arthritis, it may have some suppressive effect on the development of the disease in specific-pathogen-free and conventional F344 rats, possibly through modulation of the immune response. Adjuvant arthritis (AA) can be induced by a single injection of a water-in-oil emulsion containing mycobacterium (14), many other bacteria, and their cell walls (8, 11). A common factor among these bacterial cell walls responsible for promoting the development of AA was proved to be peptidoglycans (PGs) (12), which are found universally in all bacterial cell walls. This disease has been generally believed to be the result of a delayed-type hypersensitivity (DTH) response to components of bacterial cell walls (20), such as PGs (12). The precise mechanisms which underlie its development are still obscure. There