Serological assays may help to identify animals in advanced stages of bovine tuberculosis, but most of the tests available have suboptimal sensitivities and specificities. This study was designed to determine whether the antibody responses to defined antigens (rMPB70, rMPB64 and rMPB59) of Mycobacterium bovis at the immunoglobulin subclass level could be used to develop improved serological tests. In experimentally infected cattle it was found that the predominant serum antibody response was to rMPB70, and that an IgG1 response to this antigen was boosted strongly by skin testing. Studies in naturally infected cattle suggested that this memory IgG1 anti-rMPB70 response may be able to differentiate between skin test-reactor animals with and without lesions by comparing the ratio of the antibody response before and after skin testing. The study has provided a clearer understanding of the kinetics of antibody responses to defined mycobacterial antigens at the subclass level in bovine tuberculosis and has made it possible to develop a novel ELISA system which may be useful in disease diagnosis.