Purpose: To investigate the pharmacokinetics of intravitreally administered methotrexate. Materials and Methods: Twenty-one New Zealand white rabbits were used in the study. The pharmacokinetics of intravitreally injected 800 mu g/0.1 ml of methotrexate was investigated. Intravitreal concentration of the drug was measured at seven different times, in six eyes at each occasion, on a total of 42 eyes of 21 rabbits from a period of 30 minutes to 72 hours. Results: The volume of distribution was calculated as 1.33 ml following intravitreal injection of 800 mu g methotrexate. Vitreous concentrations of the drug were found to be decreasing related to the specific mathematical equation; drug concentration=1426.73 e(-0.1182(time)) and remained over effective dose by 81 hours with a half life of 5.9 hours. Conclusions: These findings evidenced those vitreous levels of methotrexate at various time intervals after 800 mu g intravitreal injections which formulated a mathematical equation for calculation of vitreous level of the drug at each hour.