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The objective of this study was to determine the disposition of ampicillin in plasma, uterine tissue, lochial fluid, and milk of postpartum dairy cattle. Ampicillin trihydrate was administered by intramuscular (i.m.) injection at a dose of 11 mg/kg of body weight every 24 h (n = 6, total of 3 doses) or every 12 h (n = 6, total of 5 doses) for 3 days. Concentrations of ampicillin were measured in plasma, uterine tissue, lochial fluid, and milk using HPLC with ultraviolet absorption. Quantifiable ampicillin concentrations were found in plasma, milk, and lochial fluid of all cattle within 30 min, 4 h, and 4 h of administration of ampicillin trihydrate, respectively. There was no significant effect of dosing interval (every 12 vs. every 24 h) and no significant interactions between dosing interval and sampling site on the pharmacokinetic variable measured or calculated. Median peak ampicillin concentration at steady-state was significantly higher in lochial fluid (5.27 μg/mL after q 24 h dosing) than other body fluids or tissues and significantly higher in plasma (3.11 μg/mL) compared to milk (0.49 μg/mL) or endometrial tissue (1.55 μg/mL). Ampicillin trihydrate administered once daily by the i.m. route at the label dose of 11 mg/kg of body weight achieves therapeutic concentrations in the milk, lochial fluid, and endometrial tissue of healthy postpartum dairy cattle., (© 2014 John Wiley & Sons Ltd.)