In order to assess the presence of tuberculosis in Pleistocene bison and the origin of tuberculosis in North America, 2 separate DNA extractions were performed by 2 separate laboratories on samples from the metacarpal of an extinct long-horned bison that was radiocarbon dated at 17,870+/-230 years before present and that had pathological changes suggestive of tuberculosis. Polymerase chain reaction amplification isolated fragments of tuberculosis DNA, which were sequenced, and on which spoligotyping was also performed to help determine its relationship to the various members of the Mycobacterium tuberculosis complex. Extensive precautions against contamination with modern M. tuberculosis complex DNA were employed, including analysis of paleontologic and modern specimens in 2 geographically separate laboratories.