Dietary vitamin A and beta-carotene were assessed on their interaction with lactational status to influence neutrophil function in vitro. Cows were fed 1) 53,000 IU or 2) 213,000 IU vitamin A, or 3) 53,000 IU vitamin A plus 400 mg beta-carotene/cow per d from 6 wk before to 2 wk after dry off. Blood neutrophils were isolated the day of dry off and 2 wk after dry off and incubated with retinol, retinoic acid, or beta-carotene. Phagocytosis and kill of Staphylococcus aureus were measured. Across all treatments, kill was higher after dry off than before dry off. Phagocytosis tended to be lower after dry off than before in cows fed vitamin A only. In vitro, 10(-6) M beta-carotene stimulated phagocytosis after dry off and kill before dry off in cows fed vitamin A only. In general, retinol and retinoic acid suppressed phagocytosis but did not affect kill. Neutrophils from cows fed high amounts of vitamin A were more susceptible to in vitro suppression than those from cows fed adequate amounts of vitamin A. Therefore, vitamin A and beta-carotene supplementation interacts with lactational status to influence the responsiveness of bovine neutrophils to vitamin challenge in vitro.