OBJECTIVE: To develop an alternative method for prolonged middle ear ventilation using topical mitomycin C. STUDY DESIGN AND SETTING: Twenty guinea pigs with normal ears had bilateral myringotomies performed using the argon laser. After myringotomy, either mitomycin C (0.4 mg/mL) or saline pledgets were applied topically. Monitoring consisted of otomicroscopy and distortion-product otoacoustic emissions. RESULTS: Before myringotomy, all tympanic membranes were intact, and distortion-product otoacoustic emissions were measurable. After myringotomy, none (0%) of the saline-treated myringotomies were patent at day 7 as compared with 100% of the mitomycin C-treated myringotomies. At day 42, 10 (52.6%) of 19 mitomycin-treated myringotomies remained patent and 4 (28.6%) of 14 were patent at 131 days. Five (13.1%) ears developed purulent otorrhea; 3 were mitomycin C-treated and 2 were treated with saline solution. Distortion-product otoacoustic emissions testing did not document any evidence of ototoxicity. CONCLUSION: Topical mitomycin C appears to be safe and effective at prolonging the duration of myringotomy patency in the guinea pig. SIGNIFICANCE: Mitomycin C may be useful as an adjunct for preventing myringotomy closure.