Paratuberculosis in cattle is a chronic debilitating infectious disease caused by Mycobacterium paratuberculosis. Control of paratuberculosis is based on tests that principally detect advanced stages of infections: faecal culture and serology. Tests measuring cell-mediated immunity (CMI) could improve control of paratuberculosis if able to diagnose mycobacterial infections earlier, before animals become infectious. A drawback of CMI tests for paratuberculosis has been a reported low specificity. This study re-examined CMI specificity and factors that may affect it. The specificities of the Johnin skin test and its in vitro equivalent, the gamma interferon (IFNgamma) assay, were estimated in 35 uninfected dairy herds. In each herd a random sample of 20 young (6-24 months old) and 20 adult (> or =24 months old) female dairy cattle were tested by both tests simultaneously. Skin test specificity was 93.5% using a skin thickness increase of > or =4mm as the cut-off value. IFNgamma assay specificity when interpreted using a newly developed algorithm was 93.6%. When interpreted according to two alternative algorithms provided by the IFNgamma kit suppliers, the assay had specificities of 66.1 and 67.0%. If the skin test and IFNgamma assay were used in parallel, and only animals positive on both tests were considered as M. paratuberculosis-infected, the specificity was 97.6%. Agreement between skin test and IFNgamma assay on 1631 total animals was fair (kappa=0.41). Antigen batch influenced the specificity of both the skin test, ranging from 92 to 95%, and the IFNgamma assay, ranging from 71 to 99% among batches. Test specificity also varied among herds ranging from 58 to 100% for the skin test and 57 to 100% for the IFNgamma assay. While factors affecting CMI test specificity and agreement need further evaluation, the high specificity and general agreement among these CMI tests, coupled with the excellent results obtained in the control of bovine tuberculosis using CMI tests, support their potential value in the early diagnosis and control of paratuberculosis.