We have reported that clinically isolated Prevotella nigrescens strain 22 (strain 22) produced large amounts of exopolysaccharide (EPS) and that this EPS might play an important role as a virulent factor. However, EPS production decreaces with repeated subcultures. In this study, we attempted to establish a method of animal passage that maintains the EPS production of strain 22 at high levels. We also examined the protein expression patterns of strain 22 before and after animal passage using 2-D electrophoresis. EPS production of strain 22 increased after the first animal passage, and continued to increase until the third passage. Mesh-like structures around the cells became significantly denser after animal passage. N-terminal sequence analysis of proteins revealed that three conspicuously up-regulated spots on a 2-D gel after animal passage were the homologs of bacterial heat shock proteins : chaperonin protein DnaK (HSP 70), 60kDa chaperonin (GroEL), and 10kDa chaperonin (GroES). We concluded that this method of animal passage could be used to maintain EPS production of P. nigrescens at a high level, and that the bacterial heat shock protein homologs up-regulated by the animal passage might be involved in a regulatory pathway of EPS production in P. nigrescens under stressful conditions.