The proportion of reactors to a commercial enzyme linked immunosorbent assay (ELISA) for bovine Johne's disease that were confirmed infected with Johne's disease by histology and tissue culture was determined. ELISA positive dairy cattle from the Echuca district of northern Victoria, were slaughtered at an abattoir where a standard range of specimens (mesenteric and ileocaecal lymph nodes, sections of jejunum, ileum, ileocaecal valve and colon) were collected for histology and tissue culture. Only if samples were histologically negative, were further samples submitted for tissue culture. The diagnosis was positive if histologically there were acid fast organisms with the morphology of M. paratuberculosis in typical granulomatous lesions of Johne's disease or M. paratuberculosis was detected on BACTEC culture of the tissues. Confirmation rates increased from 70.4% in 1996 to 89.4% in 2001. This is mainly because more reactors were confirmed positive by tissue culture each year as laboratory techniques improved, the proportion increasing from 0% in 1997 to 27.5% in 2000 but reducing to 16.7% in 2001. There were no significant differences between the age groups in the proportion confirmed infected. Confirmation rates were high and supported a high specificity of >99% for the ELISA when used under field conditions. The high confirmation rates support the program policy of only following up ELISA reactors at slaughter when individual herds are thought to be approaching eradication.