ACHN-490 is an aminoglycoside with activity against multidrug-resistant pathogens, including those resistant to currently used aminoglycosides. Two randomized, double-blind, placebo-controlled clinical studies investigated the pharmacokinetics (PK), safety, and tolerability of ACHN-490 injection in healthy subjects. Study 1 used a parallel-group design with escalating single (SD) and multiple doses (MD). Study 2 explored a longer duration of the highest dose tolerated in the first study. Subjects were randomly assigned to receive either ACHN-490 injection or a placebo administered by a 10-min intravenous infusion. Study 1 enrolled 39 subjects (30 active and 9 placebo) and consisted of a single dose of 1 mg/kg body weight followed by ascending SD and MD cohorts of 4, 7, 11, and 15 mg/kg for 10, 10, 5, and 3 days, respectively. Study 2 enrolled 8 subjects (6 active and 2 placebo) who received 15 mg/kg for 5 days. Safety was assessed from adverse event (AE) reporting, standard clinical laboratory procedures, and testing for renal, cochlear, and vestibular function. ACHN-490 exhibited linear and dose-proportional PK, with agreement between the studies for PK parameters assessed. The 15-mg/kg dose did not accumulate with repeated dosing over 5 days. Mean steady-state (±standard deviation) area under the concentration-time curve from 0 to 24 h (AUC(0-24)), maximum concentration of drug in serum (C(max)), half-life (t(1/2)), clearance, and volume of distribution at steady state (V(ss)) for the 15-mg/kg, day 5 dose were 239 ± 45 h·mg/liter, 113 ± 17 mg/liter, 3 ± 0.3 h, 1.1 ± 0.1 ml/min/kg, and 0.24 ± 0.04 liters/kg, respectively. AEs were mild to moderate and rapidly resolved. No evidence of nephrotoxicity or ototoxicity was observed.