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Background and objective Conventional methods to treat intraocular diseases are invasive or associated with adverse effects. A minimally invasive means of sustained-release drug delivery to the vitreous is required. This study evaluated a novel device for subtenon drug delivery to the vitreous, relative to a single subconjunctival injection. Methods Sixty adult New Zealand White rabbits were randomly assigned to receive demethylvancomycin ( DMV) by continuous subtenon delivery with the flow rate of 0.1 ml/hr for 24 hr, or as a single 0.3 ml subconjunctival injection in the right eyes. Rabbits were killed in subgroups of six at 1, 3, 6, 12 and 24 hr. The DMV concentration of the vitreous humour of the right eye was analysed by high-performance liquid chromatography. Results Overall, the vitreous DMV concentration of the subtenon group was significantly higher than that of the subconjunctival group ( F = 25.928, p = 0.001). The DMV concentration of the subtenon group was also significantly higher than that of the subconjunctival group at 3, 6, 12 and 24 hr ( t = 2.457, 5.064, 3.085, 4.207; p = 0.04, 0.01, 0.018, 0.004, respectively). In the subtenon group, the DMV concentration reached maximum (2.41 ± 0.67 μg/ml) at 6 hr, and at 24 hr was 2.37 ± 1.23 μg/ml. In the subconjunctival group, the DMV concentration reached maximum (0.48 ± 0.27 μg/ml) at 1 hr and declined to 0.09 ± 0.05 μg/ml at 24 hr. Conclusion Subtenon application with this novel minimally invasive design is an effective method for delivering an appropriate drug to the vitreous in a sustained and controllable amount. [ABSTRACT FROM AUTHOR]