Cross-reactive responses elicited by exposure to nontuberculous mycobacteria often confound the interpretation of antemortem tests for Mycobacterium bovis infection of cattle. The use of specific proteins (e.g., ESAT-6, CFP-10, and MPB83), however, generally enhances the specificity of bovine tuberculosis tests. While genes for these proteins are absent from many nontuberculous mycobacteria, they are present in M. kansasii. Mycobacterium bovis, a member of the M. tuberculosis complex, has a wide host range, is infectious to humans, and is the most common cause of tuberculosis (TB) in cattle. The presence of wildlife reservoirs (e.g., various deer species in the United Kingdom and the United States, the Eurasian badger in the United Kingdom, and brush-tailed possums in New Zealand) hinder efforts to eradicate TB within cattle and captive deer herds in developed countries. In Africa, bovine TB is rapidly spreading through Cape buffalo populations as well as other hoofstock, nonhuman primates, and various mammalian predators (23). Most notably, up to 90% of lions in areas of TB endemicity are M. bovis infected, likely due to infection rate amplification from predation on infected prey. In humans, recent TB outbreaks in several U.S. cities are linked with the ingestion of M. bovis-infected, nonpasteurized cheese from Mexico (47). Regardless of the host, the implications of TB diagnosis include regulatory action, public health concerns, movement restriction, isolation of affected individuals, and serious health issues, thus emphasizing the need for accurate diagnosis. The tests most widely used for the detection of TB in humans and cattle include the measurement of delayed-type hypersensitivity (i.e., skin testing) to purified protein derivative (PPD) and an in vitro assay for gamma interferon (IFN-␥) concentrations produced in response to PPD stimulation (Quantiferon; Cellestis, Inc., Valencia, Calif.; and Bovigam; Pfizer Animal Health, Kalamazoo, Mich.). A major limitation of PPD-based tests is cross-reactivity due to responses induced upon exposure to related bacteria, principally other nontuberculous "environmental" mycobacteria.Mycobacterium kansasii is not included in the M. tuberculosis complex, yet it may cause disease in otherwise healthy individuals, albeit infrequently, that is indistinguishable clinically from M. tuberculosis infection (2,3,14,15). A culture positive for M. kansasii from humans is not indicative of disease, as the organism may be isolated from healthy individuals, and human-to-human transmission is not documented (1). A relatively high rate of human infection has been reported in certain locales within the United States, central Europe, southeast United Kingdom, and southern part of The Netherlands, potentially associated with heavy air pollution (1, 2). As with humans, M. kansasii infection of cattle is exceedingly rare and often associated with lesions of the respiratory tract and associated lymph nodes, diagnosed postmortem (B. Harris, personal observations). Disease management of M. k...