OBJECTIVE To determine the median time to maximum concentration (tmax) of amikacin in the synovial fluid of the tarsocrural joint following IV regional limb perfusion (IVRLP) of the drug in a saphenous vein of horses. ANIMALS 7 healthy adult horses. PROCEDURES With each horse sedated and restrained in a standing position, a 10-cm-wide Esmarch tourniquet was applied to a randomly selected hind limb 10 cm proximal to the point of the tarsus. Amikacin sulfate (2 g diluted with saline [0.9% NaCl] solution to a volume of 60 mL) was instilled in the saphenous vein over 3 minutes with a peristaltic pump. Tarsocrural synovial fluid samples were collected at 5, 10, 15, 20, 25, and 30 minutes after completion of IVRLP. The tourniquet was removed after collection of the last sample. Amikacin concentration was quantified by a fluorescence polarization immunoassay. Median maximum amikacin concentration and tmax were determined. RESULTS 1 horse was excluded from analysis because an insufficient volume of synovial fluid for evaluation was obtained at multiple times. The median maximum synovial fluid amikacin concentration was 450.5 μg/mL (range, 304.7 to 930.7 μg/mL), and median tmax was 25 minutes (range, 20 to 30 minutes). All horses had synovial fluid amikacin concentrations ≥ 160 μg/mL (therapeutic concentration for common equine pathogens) at 20 minutes after IVRLP. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that, in healthy horses, maintaining the tourniquet for 20 minutes after IVRLP of amikacin in a saphenous vein was sufficient to achieve therapeutic concentrations of amikacin in the tarsocrural joint.