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In certain situations, an alternate route for parenteral drug administration in horses may be useful. The intra-osseous (IO) route may provide a safe alternative to the intravenous (i.v.) route for administration of sedatives to horses when the i.v. route is inaccessible or undesirable. Six adult horses were administered xylazine i.v. or IO in a block-randomized crossover design. For the i.v. trial, both jugular veins were catheterized, and one was used for xylazine administration, while the other was used for blood collection. For the IO trial, one jugular vein was catheterized for blood collection and an intra-osseous device was placed in the tuber coxae using a powered driver for xylazine administration. Heart rate, respiratory rate, and head position were measured, and concentration of sedation was assessed at various times up to 90 min. Xylazine concentrations were measured using high-performance liquid chromatography and noncompartmental analysis was performed. General linear mixed modeling and Wilcoxon signed-rank tests were used for statistical analysis, with  0.05. There were no significant differences in heart rate, respiratory rate, head position, concentration of sedation, Cmax, Tmax, half-life, or AUC between the i.v. and the IO routes of drug administration. No complications were observed following placement of the intra-osseous device. Intra-osseous xylazine administration provides a useful option in emergent and other settings in which i.v. access is difficult or contraindicated.