AIM To characterise the pharmacokinetics of triamcinolone acetonide (TA) in various ocular tissues following a single sub-Tenon injection. METHODS Twenty-one Chinchilla adult pigmented rabbits received sub-Tenon injection of TA (40 mg in 0.4 ml) in their right eyes. Three animals were killed at each designated time points (3 h, 1 day, 3 days, 7 days, 14 days, 21 days and 30 days) and the globes were snap frozen and dissected into aqueous, iris-ciliary body, vitreous, neuroretina and retinal pigment epithelium (RPE)/choroid. The concentrations of TA in the various ocular tissues were analysed using ultra-performance liquid chromatography, coupled with tandem mass spectrometric detection. RESULTS TA concentration followed a mono-exponential decrease over the study period in all ocular tissues of the injected eyes. The concentration was much higher in the RPE/choroid (892.14+/-558.11 ng/g at post-injection day 30) than in the other tissues (171.65+/-136.40 ng/g in neuroretina, 15.65+/-23.06 ng/ml in vitreous, 3.76+/-1.79 ng/g in iris-ciliary body, 2.64+/-0.96 ng/ml in aqueous at post-injection day 30). The TA level in the RPE/choroid had the lowest coefficient of logarithmic regression (0.07 in RPE/choroid, 0.10 in neuroretina, 0.11 in vitreous, 0.17 in iris-ciliary body, 0.18 in aqueous), indicating a 2.6 times slower clearance than in aqueous. The half-life of TA was 10.4 days in RPE/choroid. TA was detectable in the fellow eyes and was also detectable at very low levels in all blood samples during the entire study period. CONCLUSION TA was mostly cleared from RPE/choroid and retina in a mono-exponential mode. TA was above the therapeutic level for at least 30 days following a sub-Tenon injection.