White-tailed deer are significant wildlife reservoirs of Mycobacterium bovis for cattle, predators, and, potentially, humans. Infection of cattle with M. bovis stimulates an antigen-specific T-cell response, with both CD4(+) and CD8(+) cells implicated in protective immunity. Few studies, however, have examined lymphocyte subset responses to experimental M. bovis infection of white-tailed deer. In this study, a flow cytometric proliferation assay was used to determine the relative contribution of individual peripheral blood mononuclear cell subsets of M. bovis-infected white-tailed deer in the recall response to M. bovis antigen. Naive deer were challenged with M. bovis by cohabitation with infected deer. These M. bovis-challenged deer developed significant in vivo (delayed-type hypersensitivity) and in vitro (proliferative) responses to M. bovis purified protein derivative (PPD). At necropsy, typical tuberculous lesions containing M. bovis were detected within lungs and lung-associated lymph nodes of infected deer. The predominant subset of lymphocytes that proliferated in response to in vitro stimulation with PPD was the CD4(+) subset. Minimal proliferative responses were detected from CD8(+), gamma delta TCR(+), and B-cells. Addition of monoclonal antibodies specific for MHC II antigens, but not MHC I or CD1 antigens, abrogated the proliferative response. Together, these findings indicate that while CD4(+) cells from infected deer proliferate in the recall response to M. bovis antigens, this response is not sufficient to clear M. bovis and immunologic intervention may require stimulation of alternate subsets of lymphocytes.