The control of paratuberculosis depends on early identification of animals shedding bacteria, especially those animals not showing any clinical signs of disease. Clinically healthy cows older than two years were examined by cultivation of feces combined with ELISA and PCR. The feces samples were decontaminated and cultivated on Lowenstein-Jensen (L-J) with mycobactin as well as in liquid Dubos with mycobactin. The samples cultivated in Dubos were analyzed biweekly by culture on L-J, ELISA and PCR. The L-J were read once a week for 14 weeks. For ELISA and PCR and Dubos samples were spun down and the mycobacteria-pellet were lysed by bead beating with zirconium beads. DNA was amplified by PCR using primers specific for IS900. By ELISA antigen was detected with a monoclonal antibody raised against M. paratuberculosis. Of 14 animals shedding bacteria 6 were detected by primary cultivation on L-J, while a further 8 animals were detected following cultivation in Dubos. Of the 14 animals shedding M. paratuberculosis only 3 (shedding an innumerable amount of bacilli) were detected by PCR and ELISA. These results indicates that a prior cultivation in liquid medium could enhance the sensitivity of cultivation. This combined with an improved PCR technique will be a powerful diagnostic tool.