The effect of repeated infusions of Escherichia coli endotoxin on the acute phase response in the bovine mammary gland was assessed through the concentrations of tumor necrosis factor alpha (TNF-alpha) in milk. Four clinically normal lactating cows received two intramammary infusions of E coli endotoxin (33 micrograms) 24 h apart in the same mammary quarter. Along with the second infusion, the cows received one dose of endotoxin in the contralateral quarter. Milk was collected at varying intervals before and after infusion and TNF-alpha concentrations were determined by ELISA. Following the first infusion at 0 h, the mean concentrations of TNF-alpha augmented from undetectable concentrations to a maximum of 0.4 ng/mL at 4 h and declined to below 0.04 ng/mL at 24 h, the time of the second infusion. In the quarters challenged twice, the increase in TNF-alpha concentrations was abrupt, culminating at 11.7 ng/mL 6 h later (at 30 h). The increases in TNF-alpha concentrations were similar in the contralateral quarters infused once. TNF-alpha concentrations in the control, uninfused quarters of infused cows remained undetectable (< 0.04 ng/mL). Despite the low TNF-alpha response following the first infusion, mean somatic cell counts increased markedly, being only slightly lower than after the second infusion (10(7)/mL and 5 x 10(7)/mL at 8 h and 32 h, respectively) in the quarters challenged twice. After the first infusion, none of the cows developed fever, but following the second infusion, rectal temperature increased markedly, culminating 6 h after the second infusion. These results show that an infusion in one quarter of an amount of endotoxin sufficient to induce a pronounced cell recruitment but insufficient to induce a marked TNF-alpha secretion following the first infusion sensitized not only that quarter but also the contralateral one to a second infusion with the endotoxin. It is thus possible that sensitization of the whole udder follows a first contact with a moderate dose of endotoxin in one quarter.