A high performance liquid chromatography (HPLC) method was developed, validated and used to analyse haptoglobin concentrations in serum samples taken from eight cows which had been challenged twice intramammarily with Escherichia coli. The results of the HPLC were compared with those from a photometric assay. The kinetics of the haptoglobin response were analysed with pharmacokinetic computer software. In contrast with the photometric assay, the HPLC was sufficiently sensitive to detect normal background levels of bovine serum haptoglobin. The serum haptoglobin concentrations of healthy cows ranged from 22 to 47 mg litre-1. As the concentration of haptoglobin increased, the results of the two methods correlated well (r = 0.96). A 52-fold increase in serum haptoglobin was detected after the first challenge with E coli. The mean pharmacokinetic parameters of the response after the first challenge were: lag phase 12 hours, t2/1increase 20 hours, tmax 72 hours, t1/2decrease 46 hours, and mean residence time 112 hours. The second challenge three weeks later resulted in a significantly lower haptoglobin response, the area under curve being 35 per cent of that after the first challenge. The clinical signs and inflammatory changes in the milk did not differ significantly between the challenges.