After immunization of four calves with a live modified Mycobacterium paratuberculosis vaccine the course of the humoral and cell mediated immune reactions was studied during a 2-year clinical investigation. Furthermore, the possibility of shedding of the vaccine strain and the influence of the vaccination on the tuberculin skin test was determined. In addition to standard procedures recently developed diagnostic methods (antibody enzyme-linked immunosorbent assay, interferon-gamma test, polymerase chain reaction) were used. A cell-mediated immune reaction, reflected in an increased, specifically induced, interferon-gamma production developed much earlier (1-2 weeks post-immunization) than humoral immunity (8-16 weeks post-gamma immunization). While the increase in antibody titres was transient, declining to extremely low levels 48-60 weeks post-immunization, cell-mediated immunity remained detectable until the end of the investigation. Spread of the vaccine strain into the body and shedding were never detected during the whole course of the study except for one colon site in one calf. As late as 2 years after vaccine application positive or doubtful skin reactions against M. bovis purified protein derivative were measured, reflecting possible interference of the immunization with the diagnosis of bovine tuberculosis. At the end of the investigation, a positive cell-mediated immune reaction was detected the control animal although clinical, pathological and bacteriological examinations gave no indication for a mycobacterial infection.