The BACTEC MGIT 960 system was evaluated for the growth and detection of Mycobacterium avium subspecies paratuberculosis. The isolation and relative enumeration of M. paratuberculosis shed in the manure of ruminants is indicative of the presence and onset of active Johnes disease. Currently, the culture of M. paratuberculosis from manure is performed using a variety of solid media including LJ slants and Herrold's Egg Yolk agar. This evaluation is concerned with optimizing a Middlebrook based formulation for the growth and detection of M. paratuberculosis using the BACTEC 960 instrument. The basal Middlebrook medium was modified to evaluate the addition of various peptones, bovine serum albumen, mineral salts and osmolarity modifications. The tests included at least three strains of M. paratuberculosis, as determined by strict mycobactin-dependent growth, as well as other strains of M. avium. Mycobacterium tuberculosis was also included in media evaluations. The results show that the basic Middlebrook formulation typically used to grow and detect M. tuberculosis is not optimized for the growth and recovery of M. paratuberculosis. The data indicates that careful selection of BSA and the addition of certain peptones stimulates the growth and metabolism of M. paratuberculosis. This will greatly enhance the potential for using the BACTEC MGIT 960 system as a fully automated diagnostic tool for Johnes disease in ruminants.