During bacterial infection of the bovine mammary gland, large numbers of leukocytes migrate into the udder, resulting in the establishment of a host response against the pathogen. Currently, the specific leukocyte populations mediating this immune response are not well defined. In the studies described here, we analyzed blood and milk from healthy cows and cows with naturally occurring mastitis to determine if distinct and T-lymphocyte subsets were involved in the response of the udder to a mastitis pathogen and if the type of mastitis pathogen influenced the subset composition of these responding leukocytes. Although blood samples from cows with confirmed staphylococcal and streptococcal mastitis were characterized by increased numbers of T cells, the most dramatic changes in leukocyte distributions occurred in milk samples from these cows, with a 75% increase in T-cell levels and a 100% increase in T-cell levels relative to the levels in milk samples from healthy animals. Interestingly, the increase in T-cell numbers observed in milk from cows with staphylococcal mastitis was primarily due to increased numbers of CD4⁺ T cells, while the increase in T-cell numbers observed in cows with streptococcal mastitis was due to a parallel increase in both CD4⁺ and CD8⁺ T-cell numbers. The increased numbers of T cells in milk from cows with staphylococcal and streptococcal mastitis were due to a selective recruitment of a distinct T-cell subset (GD3.1⁺), while no change in the numbers of GD197⁺ T cells was observed. We also analyzed adhesion protein expression on blood and milk leukocytes and found that, in comparison to the situation for healthy cows, L-selectin was down-regulated and CD18 was up-regulated on leukocytes from cows with mastitis. Thus, shedding of L-selectin and up-regulation of CD18 by neutrophils may provide a sensitive indicator of early inflammatory responses during bovine mastitis. Overall, these studies suggest that distinct and T-cell subsets are involved in the host defense of the udder against mastitis infection and that selective recruitment of these T-cell subsets depends on the infectious agent involved.