Technical Abstract: White-tailed deer (Odocoileus virginianus) have recently emerged as a source of Mycobacterium bovis infection for cattle within North America. The objective of this study was to evaluate the immunoglobulin reactivity to crude mycobacterial antigens of serum obtained from M. bovis-infected deer. Deer were experimentally inoculated with M. bovis strain 1315 either by intratonsilar instillation or by exposure to M. bovis-infected (i.e., in contact) deer. To determine the temporal kinetics of the response including affects of antigen administration for comparative cervical skin testing, serum was collected periodically and evaluated by ELISA for immunoglobulin (i.e., IgG heavy and light chains) reactivity to mycobacterial antigens. Reactivity to M. bovis purified protein derivative (PPDb) exceeded (p < 0.05) the reactivity to M. avium PPD (PPDa) only after in vivo administration of PPDa and PPDb for comparative cervical testing of infected deer. The mean immunoglobulin response, as measured by ELISA, of intratonsilar-inoculated deer to a proteinase k-digested whole cell sonicate of M. bovis strain 1315 exceeded (p < 0.05) that of pre-challenge responses to this antigen at approximately 1 month post inoculation and throughout the remainder of the study (i.e., ~ 11 months). This response also exceeded (p < 0.05) that of non-infected deer. While encouraging, further studies are necessary to validate the usage of proteinase k-digested M. bovis antigens in antibody-based assays of tuberculosis.