Murine antibody against Mycoplasma pulmonis (Mp) was detected sensitively and specifically in experimentally and naturally infected animals by an enzyme-linked immunosorbent assay (ELISA), using urease conjugated antimurine immunoglobulin. More than 98% of the experimentally infected mice and rats exhibited positive reaction in the ELISA two or more weeks after infection, and the titer remained for a prolonged period (up to one year) after infection. However, we failed to detect antibody in the sera of one-week-postinfected animals. Mice and rats from breeding colonies were tested with the ELISA and compared with isolation of Mp from the respiratory organs. Positive reactions were shown in the ELISA using the sera from 91% of the mice and 98% of the rats from which the organisms were isolated. Conversely, 97% of the mice and 78% of the rats among Mp-free animals showed negative results in the ELISA. The sensitivity and specificity of the complement fixation test, which has been used widely for serodiagnosis of Mp-infection, were apparently lower compared to those of the ELISA. From these results, the ELISA was found to be available for the serodiagnosis of Mp-infection in mice and rats.