Technical Abstract: Mycobacterium bovis infection of cattle represents a natural host/pathogen interaction and, in addition to its economic and zoonotic impact, represents a model for human tuberculosis. Extravasation and trafficking of activated lymphocytes to inflammatory sites is modulated by differential expression of multiple surface adhesion molecules. However, effects of M. bovis infection on adhesion molecule expression are not characterized. To determine these changes, peripheral blood mononuclear cells (PBMC) from M. bovis-infected cattle were stimulated with M. bovis purified protein derivative (PPD) or pokeweed mitogen (PWM) and evaluated concurrently for proliferation and activation marker expression. Stimulation with PPD or PWM increased CD25 and CD44 mean fluorescence intensity (mfi) and decreased CD62L mfi on CD4+ cells from infected animals. CD62L mfi on PPD- and PWM-stimulated gammadelta TCR+ and CD8+ cells was also reduced when compared to that of non-stimulated gammadelta TCR+ and CD8+ cells. Using a flow cytometric-based proliferation assay, it was determined that proliferating cells, regardless of lymphocyte subset, exhibited increased expression of CD25 and CD44 and decreased expression of CD62L when compared to that of cells that had not proliferated. In contrast to proliferation, activation-induced apoptosis of CD4+ cells resulted in a significant down regulation of CD44 expression. Lymphocytes obtained from lungs of M. bovis-infected cattle also had reduced expression of CD44 when compared to lymphocytes from lungs of non-infected cattle. These alterations in surface molecule expression upon activation likely impact trafficking to sites of inflammation and the functional capacity of these cells within tuberculous granulomas.