The ocular pharmacokinetics of thiamphenicol (TAP, CAS 15318-45-3) was studied in rabbits by means of the assessment of its ocular and systemic absorption, and urinary excretion after instillation of 0.5% TAP eye drops. TAP concentrations in aqueous humor, plasma and urine were evaluated by a coupled LC/GC method (detection limit = 0.1 ng/ml), because the necessity to have a technique much more sensitive than the traditional chromatographic ones available in order to quantify the very low drug concentrations in biological fluids produced by the ocular treatment, and generally by a topical administration. The intravenous route was chosen as reference and allowed the absolute bioavailability to be estimated. TAP proved to be well absorbed through the cornea with the peak aqueous humor concentration of 110 ng/ml at 45 min following the instillation. The good ocular absorption of TAP was confirmed by the plasma concentrations observed after instillation of 0.5% eye drops. In any case, these concentrations were more than 1000 times lower than those observed after the intravenous treatment at the dose normally used for infectious diseases, allowing to exclude any systemic toxicity of TAP eye drops. The absolute ocular bioavailability was 16.2% when estimated from the AUC values and 34.0% from the cumulative urinary excretion values.