Background Infection after ACL reconstruction is uncommon but catastrophic. Prophylactic graft saturation in vancomycin reportedly reduces infection rates. Questions/purposes We characterized vancomycin elution from soaked tendons. Specifically, the effect of rinsing was studied. We also determined how vancomycin concentration in the soak solution and tendon dimension influenced this elution rate, and examined whether the vancomycin amount released was lower than osteoblast and chondroblast toxic concentrations. Methods Bovine tendons were wrapped in sterile gauze swabs presoaked with 5-, 2.5-, or 1.25-mg/mL vancomycin solutions. After 10 minutes, rinsed and unrinsed tendons were placed in 100 mL agitated 37àphosphate-buffered saline (PBS). One-milliliter samples taken at 10 minutes and 1, 6, 12, 24, and 72 hours were analyzed by high-performance liquid chromatography. Results The maximum elution rate occurred between 10 minutes and 1 hour, with no lag between experiment initiation and drug appearance in the solution. Rinsing affected the initial amount in solution but had little influence on drug release after 10 minutes. Vancomycin diffusion rates were dependent on soak solution concentration at all sampling intervals. The vancomycin amount released or eluted did not increase after the 1-hour interval. At 24 hours, concentrations were 45 ᠱ2, 16 ᠱ, and 9 ᠳ 姯mL for the 5-, 2.5-, and 1.25-mg/mL solutions, respectively. Higher elution rates were observed in larger-volume tendons. Conclusions Soaked tendon grafts can act as reservoirs for vancomycin, with the amount released and elution profile dependent on rinsing, tendon volume, and soak solution concentration. Vancomycin elution was lower than previously reported osteoblast and chondroblast toxicity concentrations and above the minimum inhibitory concentration for Staphylococcus. Clinical Relevance Presoaking ACL reconstruction autografts with vancomycin may reduce the risk of ACL reconstruction infection without the risk of local or general toxicity.