Purpose and Background: To measure azithromycin levels in rabbit lacrimal and Harder glands, conjunctiva and plasma after a single oral dose of 20 mg/kg. Drug levels in lacrimal gland tissue are significant in trachoma because the gland may be involved in the disease process and it is the source of tears by which the drug is carried to the external eye. Methods: Lacrimal and Harder glands, conjunctiva and plasma were collected from New Zealand white female rabbits at 24, 48, 72, 96 and 144 h following a single oral dose of azithromycin (20 mg/kg). Azithromycin levels in tissue and plasma were measured using high-performance liquid chromatography (HPLC) electrochemical detection. Results: Azithromycin levels peaked within the first 24 h in all tissues and plasma assayed. The highest concentration was in the lacrimal gland (6.2 µg/g, SD ± 0.8), followed by Harder gland (4.4 µg/g, SD ± 0.8), conjunctiva (0.9 µg/g, SD ± 0.5) and plasma (0.06 µg/g, SD ± 0.03). These concentrations reached their lowest measured levels at 120 and 144 h. Conclusion: Azithromycin levels measured throughout the 144 h after dosing were consistently above the minimum inhibitory range (MIC) for Chlamydia trachomatis (0.03–0.25 µg/ml) in the lacrimal glands, while the conjunctiva maintained a concentration above the MIC for 96 h and stayed within MIC levels for 144 h.