This study was conducted to determine the passage ratio of amoxicillin into milk and its pharmacokinetics in milk and plasma after intramuscular administration. Five healthy dairy cows (Holstein, weighing 450-500 kg, aged 2-4 years) were used in this study. They received single intramuscular amoxicillin at a dose of 14 mg/kg body weight. Blood and milk samples were collected prior to drug administration (0); after 15, 30, 45, 60, and 90 min; and 2, 3, 4, 6, 8, 10, and 12 hr after administration. The plasma and milk concentrations of amoxicillin were determined using high-performance liquid chromatography with ultraviolet detection. The passage ratio of amoxicillin into milk and plasma was determined using both AUC-based calculation and milk and plasma concentrations at sampling times; it was calculated 0.46 and 0.52, respectively. The terminal half-life and mean residence time of amoxicillin were 6.05 and 8.60 hr in plasma and 2.62 and 5.35 hr in milk, respectively. The C-max2 levels of amoxicillin in plasma and milk were measured as 1,096 and 457 ng/ml, respectively. It was observed that amoxicillin exhibited a secondary peak in plasma and milk. This study was the first to report on the passage ratio of amoxicillin into milk in lactating cows.