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Previous studies have shown that regional limb perfusion (RLP) using the palmar digital (PD) vein delivers therapeutic concentration of amikacin to the distal limb. Our hypothesis was that using the cephalic and saphenous veins for RLP will enable delivery of therapeutic concentrations of amikacin to the distal limb. Nineteen healthy horses participated in the study. The cephalic, saphenous, or PD vein was used to perfuse the limb with amikacin. Two grams of amikacin was used for RLP using the saphenous and the cephalic veins, and one gram was used in the PD vein. Synovial samples were collected from the metacarpo-/metatarsophalangeal (MCP/MTP) joint, and blood samples were collected from the jugular vein. Maximum concentration (Cmax) of amikacin in the MCP/MTP joint using the cephalic and the saphenous vein was 277 and 363 mg/L, respectively. The amikacin concentrations achieved in the synovial fluid of the MCP/MTP joint in the current study were between 69 and 91 times the minimally inhibitory concentration of common susceptible bacterial pathogens causing orthopedic infections in horses. To conclude, this study shows that use of the proximal veins for RLP to treat distal limb infections is a viable alternative to using the palmar or plantar digital vein.