Gepotidacin, a novel, first-in-class triazaacenaphthylene antibiotic, inhibits bacterial DNA replication by a distinct mechanism of action. We report the pharmacokinetics, safety, and tolerability of gepotidacin following single or multiple ascending doses. Studies 1 and 2 were randomized, single-blind, placebo-controlled trials in healthy adults aged 18-60 years, who received single (Study 1 [NCT02202187]; 100-3000 mg) or repeat (Study 2 [NCT01706315]; 400 mg twice daily to 2000 mg thrice daily) ascending doses of gepotidacin. Study 3 (NCT02045849) was an open-label, 3-part, study in healthy adults; here we report on part 3, a 2-period, repeat-dose, crossover study. Healthy elderly participants received repeat 1500 mg gepotidacin twice daily with or without a moderate-fat meal. Primary endpoints were pharmacokinetics (Studies 1 and 2) and safety (Studies 1 and 3 part 3). Gepotidacin pharmacokinetic parameters were comparable across all ages and were dose proportional. In all studies, gepotidacin was readily absorbed with median time to maximum concentration observed (tmax ) ranging from 1.0-4.0 h across all doses. Median apparent terminal phase half-life (t½ ) was consistent across studies and doses (range: 5.97-19.2 h). Steady state was achieved following repeated dosing for 3-5 days; gepotidacin pharmacokinetic parameters were time invariant after repeated oral dosing. A moderate-fat meal did not affect gepotidacin pharmacokinetic parameters. Gepotidacin was generally well tolerated, with no drug-related serious adverse events reported. Collectively, these pharmacokinetic and safety data across a wide range of doses in healthy participants aged ≥ 18 years support the development of gepotidacin in further clinical studies.