The ability of Staphylococcus aureus alpha-toxin to recruit neutrophils in the milk of vaccinated cows and the bactericidal efficiency of these neutrophils were evaluated. Six lactating Holstein cows that were free of intramammary infection received systemic immunization by subcutaneous injection of Freund's incomplete adjuvant with alpha-toxin (n = 2), alpha-toxin mixed with type 5 capsular polysaccharide (n = 2), or a conjugate of these two antigens (n = 2). Controls (n = 4) and vaccinated cows (n = 6) received intramammary infusions of alpha-toxin. No increase in somatic cell count was recorded in quarter milk samples from unimmunized cows; however, 10 micrograms of alpha-toxin induced a local inflammatory reaction in vaccinated cows that was characterized by early and massive cellular recruitment into the mammary gland. More than 90% of the recruited cells were neutrophils. The speed and magnitude of the cellular recruitment were dose-dependent; the threshold dose was 0.6 microgram. Milk samples showed significant bactericidal activity against the type 5 S. aureus strain, regardless of the vaccine used, and showed a decrease in bacterial count of about 2 log10 from the initial inoculum. The best efficiency was recorded during the early phase of cellular recruitment with concomitant activation of blood-derived neutrophils. This study demonstrates that a bacterial virulence factor, alpha-toxin, is able to induce immune recruitment of neutrophils for efficient bactericidal activity in milk when cows are immunized with alpha-toxin that is used either as a nonconjugate vaccinate or as a carrier protein in a conjugate vaccine. The study also suggests that neutrophils that are recruited from blood are activated during inflammation in response to specific antigens.