The plasma and synovial fluid pharmacokinetics and safety of cefquinome, a 2-amino-5-thiazolyl cephalosporin, were determined after multiple intravenous administrations in sixteen healthy horses. Cefquinome was administered to each horse through a slow i.v. injection over 20 min at 1, 2, 4, and 6 mg/kg (n = 4 horses per dose) every 12 h for 7 days (a total of 13 injections). Serial blood and synovial fluid samples were collected during the 12 h after the administration of the first and last doses and were analyzed by a high-performance liquid chromatography assay. The data were evaluated using noncompartmental pharmacokinetic analyses. The estimated plasma pharmacokinetic parameters were compared with the hypothetical minimum inhibitory concentration (MIC) values (0.125–2 μg/mL). The plasma and synovial fluid concentrations and area under the concentration–time curves (AUC) of cefquinome showed a dose-dependent increase. After a first dose of cefquinome, the ranges for the mean plasma half-life values (2.30–2.41 h), the mean residence time (1.77–2.25 h), the systemic clearance (158–241 mL/h/kg), and the volume of distribution at steady-state (355–431 mL/kg) were consistent across dose levels and similar to those observed after multiple doses. Cefquinome did not accumulate after multiple doses. Cefquinome penetrated the synovial fluid with AUCsynovial fluid/AUCplasma ratios ranging from 0.57 to 1.37 after first and thirteenth doses, respectively. Cefquinome is well tolerated, with no adverse effects. The percentage of time for which the plasma concentrations were above the MIC was >45% for bacteria, with MIC values of ≤0.25, ≤0.5, and ≤1 μg/mL after the administration of 1, 2, and 4 or 6 mg/kg doses of CFQ at 12-h intervals, respectively. Further studies are needed to determine the optimal dosage regimes in critically ill patients.