The eaeA-positive, attaching and effacing (A/E) O45 E. coli isolates from pigs express an EaeA protein with an estimated molecular weight of 97 kDa. In the present study, a monoclonal antibody was raised against the EaeA protein of an A/E O45 isolate. Cross reaction of the monoclonal antibody with the EaeA protein of A/E strain of the rabbit (RDEC-1), but not with those of A/E strains of the human (E2348/69) and dog (89-4221), was observed. Reactions of the monoclonal antibody to A/E isolates in the O45 serogroup on the ELISA varied among isolates and appeared to be correlated with in vivo A/E capacity of these isolates. The EaeA protein of A/E O45 E. coli has an apparent isoelectric point of 8.4 and is exposed on the bacterial surface. The monoclonal antibody provides a useful tool for characterization of the EaeA protein of E. coli isolates from pigs.