Three serological tests for the diagnosis ofMycobacterium bovisinfection were evaluated on 29 possums (Trichosurus vulpecula) with tuberculosis and on 100 possums from a tuberculosis-free area. An indirect ELISA usingM. bovisculture filtrate as the antigen had a sensitivity of 45% and a specificity of 96%, while an indirect ELISA using aM. bovisspecific antigen (MPB70) had a sensitivity of 21% and a specificity of 98%. A blocking ELISA which utilised a monoclonal antibody against MPB70 had a sensitivity of 28% and a specificity of 99%. Combination of the test results of the three ELISAs resulted in an increase in sensitivity to 51% and a decrease in specificity to 93%. A previous study has shown that possums experimentally infected withM. bovisproduced cellular responses toM. bovisantigens relatively early in the infection, but these responses decreased in the terminal stages of the disease. In contrast, analysis of serological responses in the current study from sequentially collected sera of possums experimentally and naturally infected withM. bovisshowed that antibodv was first detected late in the disease.