Technical Abstract: Mastitis, caused by bacterial infection of the mammary gland, is a major disease of dairy cattle. The greatest risks of intramammary infection occur at the end of lactation and at the initiation of the next lactation when the cow calves. Treating serum with zymosan (yeast cell wall preparation) causes complement to cleave, allowing this serum to serve as a source of C5a, a potent chemo-attractant and activator of the immune system. Our hypothesis was that intramammary infusion of zymosan-treated serum (ZTS) would recruit neutrophils and generate prolonged activity in lymphocytes within the mammary gland. Ultimately this could help prevent bacterial infections in cows at dry off and at the initiation of lactation. Two ipsilateral quarters of the mammary gland of each cow were infused with ZTS (12.5 mL/quarter) and two quarters with saline in 8 cows shortly after lactation ended. Mammary secretions were collected periodically throughout the dry period and the first two weeks of the next lactation. Activation status of lymphocytes and neutrophils in those secretions was assessed based on the intracellular presence or absence of IFN-gamma and IL-8 as determined by flow cytometry. ZTS infusion greatly increased neutrophil numbers in mammary secretions for the first week only. The percentage of IFN-gamma positive lymphocytes and neutrophils and the percentage of IL-8 positive neutrophils exhibited a sustained increase in secretions from ZTS treated quarters through the first two weeks of lactation (P < 0.1). ZTS can stimulate neutrophil and lymphocyte mediated immune defense mechanisms in the mammary gland, which may provide a useful means of preventing new intramammary infections during the dry period as well as at the initiation of lactation.