OBJECTIVE The aim of this study was to investigate whether the ocular pharmacokinetic parameters observed following systemic administration are also seen following topical administration. METHODS Azithromycin concentrations were measured by HPLC-MS in pigmented rabbits' tears, cornea, bulbar conjunctiva, and aqueous humor following single instillation and twice-daily instillations for three consecutive days of topical 1.50% azithromycin dihydrate solution. RESULTS Following a single administration, azithromycin levels were higher than the MIC 4 microg/g breakpoint for susceptible germs for at least 4 hr in tears, 1 hr in conjunctiva, and 1 hr in cornea after instillation. Following multiple administrations, azithromycin levels were higher than the MIC 4 microg/g for at least 16 hr in tears, 24 hr in conjunctivae, and 1 week in cornea after the last instillation. CONCLUSIONS Both dosage regimens resulted in adequate and long-lasting azithromycin levels in the conjunctiva, the ocular target tissue relative to the expected therapeutic indication in man (bacterial conjunctivitis), and also in the cornea and tears.