The objectives of this study were to investigate the pharmacokinetics of once-daily amikacin in healthy neonates, to determine amikacin concentrations in hospitalized foals, and to determine the minimum inhibitory concentrations (MICs) of amikacin against gram-negative isolates from blood cultures in septic foals. Median half-life, clearance, and volume of distribution of amikacin in healthy 2- to 3-day-old foals after administration of an intravenous bolus of amikacin (25 mg/kg) were 5.07 hours (4.86-5.45 hours), 1.82 mL/min/kg (1.35-1.97 mL/min/kg), and 0.785 L/kg (0.638-0.862 L/kg), respectively. Statistically significant (P <.05) decreases in area under the curve (14% decrease), mean residence time (19% decrease), and C24h plasma amikacin concentrations (29% decrease) occurred between days 2–3 and 10–11. Plasma amikacin concentrations in healthy foals at 0.5 hours (C05h) were significantly higher (P= .02) than those of hospitalized foals. Sepsis, prematurity, and hypoxemia did not alter amikacin concentrations. The MIC at which 90% of all gram-negative isolates from equine neonatal blood cultures were inhibited by amikacin was 4 |xg/mL, suggesting that amikacin C05h of 40 μg/mL should be targeted to achieve a maximum serum concentration to MIC ratio of 10:1. The proportion of foals with C05h >40 μg/mL was significantly higher (P <.0001) in hospitalized foals receiving a dose of amikacin at 25 mg/kg (22/24 or 92%) than in foals receiving a dose at 21 mg/kg (9/25 or 36%), whereas no difference was found in the proportion of foals with C24h concentrations ≥3 μg/mL between the 2 groups. An initial dose at 25 mg/kg is recommended for once-daily amikacin in equine neonates.