The local and systemic immune response of the lactating cow during the 10 week period after intramammary (IMM) vaccination with killed Mycobacterium bovis (M. bovis) was evaluated. Antigen (tuberculin)-reactive lymphocytes were present in the milk as early as 2 weeks post-vaccination, and in the blood at 6 weeks after vaccination. The milk lymphocytes, compared to the blood lymphocytes were consistently more responsive to tuberculin. Both blood and milk lymphocytes responded in vitro to the lectins, phytohemagglutinin-A (PHA-P) and concanavalin A (Con A), although the milk lymphocytes were consistently less responsive than the blood lymphocytes during the period. Anti-tuberculin antibody was significantly elevated in the milk and blood of the vaccinated animals at 10 weeks post-vaccination. Infusion of tuberculin into the mammary glands of the cows 10 weeks after vaccination resulted in a marked increase in the number of milk leukocytes. The influx of leukocytes initially consisted of polymorphonuclear leukocytes (PMNL), and later, mononuclear leukocytes. Intramammary vaccination also resulted in antigen recognition at sites distant from the mammary gland.