The purpose of this project was to determine the effect of dexamethasone (DEX) treatment of tuberculous cows on antigen-stimulated gamma-interferon (gamma-IFN) production in a commercial Mycobacterium bovis gamma-interferon test (gamma-IFN test) developed for diagnosis of bovine tuberculosis. In the gamma-IFN test an enzyme-linked immunosorbent assay is used to detect bovine gamma-IFN in the plasma from whole blood samples cultured with M. bovis and Mycobacterium avium tuberculin purified protein derivatives (PPDs). DEX is a synthetic glucocorticoid commonly used as a potent anti-inflammatory agent, and experimentally as a model of stress-induced immunosuppression in cattle. DEX treatment has previously been associated with decreased lymphocyte response to mitogens in cattle, which led to the current hypothesis that DEX treatment would suppress stimulated gamma-IFN production resulting in false negative results in the gamma-IFN test. In replicate studies using naturally infected dairy cows, blood was drawn daily for at least 2 days prior to DEX treatment, during 3 days of DEX treatment, and for at least 9 days post-DEX. Results of the gamma-IFN test were evaluated by optical density (OD), and by three OD calculation methods: two different methods suggested by the manufacturer, and a method adapted from the evaluation of a bovine gamma-IFN test used in Australia. Prior to DEX treatment all cows had positive gamma-IFN tests by each calculation method. As early as 24 h after the first DEX injection a decline in PPD-stimulated gamma-IFN production was reflected in OD data for all cows. Calculated gamma-IFN test results were negative after DEX treatment for all but one cow, which was known to produce relatively large amounts of gamma-IFN as measured by this test. The degree of gamma-IFN suppression, and the number of days that gamma-IFN test results were negative after DEX treatment (1-8 days), varied by cow and by data calculation method. Treatment with DEX is associated with suppressed PPD-stimulated gamma-IFN production, which may be reflected as false negative results in the gamma-IFN test depending on the data calculation method applied. The results have implications for the management conditions and medical treatment schedule under which samples for the gamma-IFN test are collected.