A group of 252 cattle without clinical signs of paratuberculosis (paraTB) in 10 herds infected with paraTB and a group of 117 cattle in 5 herds without paraTB were selected. Whole-blood samples were stimulated with bovine, avian, and johnin purified protein derivative (PPD) and examined for gamma interferon (IFN-gamma) release. For diagnosis of paraTB, satisfactory estimated specificities (95 to 99%) could be obtained by johnin PPD stimulation irrespective of interpretation relative to bovine PPD or no-antigen stimulation alone, but numbers of test positives in the infected herds varied from 64 to 112 with different interpretation criteria. For a limited Dumber of test-positive animals, no change in the test results could be observed with increasing antigen concentrations but IFN-gamma responses were significantly reduced (P <0.0001) and four out of seven reactors tested negative when stimulation was performed on day-old samples. Denmark is free of bovine tuberculosis, but cross-reactivity with paraTB could be documented for cattle more than 14 months old in paraTB-infected herds compared with those in non - paraTB-infected herds. In both paraTB-free and paraTB-infected herds, false positives were observed when the test was applied to calves less than 15 months of age. Until novel antigen formulations more specific for these diseases are available, interpretation of the IFN-γ test must be individually adjusted to fit specific needs and the context within which the test is applied and, for paraTB, the test seems most appropriate for use as a supportive tool for evaluation of disease-preventive measures in young stock.