Background: The purpose of this study is to compare the effects of two different doses of 5-fluorouracil (5-FU) on closure rates and histopathological findings in myringotomy to those of simple myringotomy with saline application. Material/Methods: 36 rats were divided into two study groups and a control group of 12 rats each. After bilateral myringotomy, we applied 5-FU 50 mg/ml in group A, 10 mg/ml in group B and saline in the control group for 10 minutes. Examination was made by otoendoscopy on days 1, 3, 5, 7, 10, 15, 20, 25. After the follow tip examination, two rats from each group were randomly sacrificed on days 7, 15, 25 and 35. Both tympanic membranes were investigated for fibrosis and inflammation by light microscopy. Results: Most of the tympanic membranes from group B and the control group healed on the 10(th) day, but 16 myringotomies were patent in group A; by the 25(th) day, all myringotomies in all three groups were completely healed. The mean healing time was 14.62 days, 10.20 days and 10.40 days respectively in groups A, B and control. Fibrosis and inflammation were less in group A than in group B and the controls. Conclusions: 5-FU at a concentration of 50 mg/mL is effective in prolonging the patency of myringotomies in rat tympanic membranes, while a concentration of 10 mg/mL has no greater effect than a simple myringotomy procedure with saline application.