An enzyme-linked immunosorbent assay (ELISA) to measure Streptobacillus moniliformis antibodies in mice and rats was developed. Twelve S. moniliformis strains originating from cases of rat-bite fever and Haverhill fever in man and from various rodent species, showed considerable serological relationship. The ELISA appeared specific since antibodies to S. moniliformis were absorbed by autologous and homologous antigen, but not by heterologous bacterial antigens. Acholeplasma laidlawii showed partial serological relationship with S. moniliformis. The ELISA was validated using experimental infections in mice and rats. These studies and observations in naturally infected feral rats, confirmed that S. moniliformis is difficult to grow on primary isolation, and that the ELISA for S. moniliformis antibodies revealed more contaminated animals than culture.