Florescence flow cytometry and monoclonal antibodies were used to analyze the composition of leukocytes from peripheral blood and mammary gland secretions. Samples were obtained from Holstein dairy cows, free of IMI, at six time points during the lactation cycle. The percentage of monocytes in peripheral blood mononuclear cells varied from 15 to 31%; the lowest percentage occurred at the early nonlactation period. The percentage of T lymphocytes from mammary gland secretions varied from 16% during the periparturient period to 62% at late lactation. The B lymphocytes varied from 7% at late lactation to 25% during the periparturient period. Macrophages varied from 21 to 69%; the highest percentage occurred during the periparturient period. The mean ratio of CD4+:CD8+ T lymphocytes in the peripheral blood and mammary gland secretions was 1.53 and .85, respectively. A subpopulation of activated CD8+ T lymphocytes present in mammary gland secretions throughout the lactation cycle coexpressed a new activation molecule, ACT2. Increases in the proportion of CD8+ T lymphocytes were associated with an increase in the percentage of CD8+, ACT2+ T lymphocytes. Activated CD8+ T lymphocytes may play an important role in the regulation and expression of the local immune response to pathogens.