Abstract Objective We sought to determine a dose-response curve for topical mitomycin C when used to prolong the patency of laser-assisted myringotomies. Study design and setting Bilateral myringotomies were performed using the argon laser in 40 guinea pigs with normal ears. Pledgets with (0.05, 0.2, 0.4, or 2.0 mg/mL) mitomycin C were applied topically. Monitoring consisted of weekly or biweekly otomicroscopy. Results As in previous studies, all saline-treated myringotomies closed by day 7. By day 14, all myringotomies (100%) in the 0.05 mg/mL group were closed. By contrast, all myringotomies (100%) remained patent in the 0.2 and 0.4 mg/mL treatment groups, and 56% of the myringotomies remained patent in the 2.0 mg/mL group at day 14. By day 56, all myringotomies were closed in the 2.0 mg/mL group, 5 (50%) myringotomies were patent in the 0.4 mg/mL group, and 1 (11%) myringotomy was patent in the 0.2 mg/mL group. The study was terminated on day 84 (12 weeks). At that time, only the 0.4 mg/mL group had patent myringotomies (n = 3; 30%). The highest dose (2.0 mg/mL) of mitomycin was significantly associated with otorrhea. Otorrhea also appeared to be associated with earlier myringotomy closure. Conclusion There is a dose-response curve for topical mitomycin C when used for prolonging myringotomy patency in doses up to 0.4 mg/mL. Higher doses do not appear to prolong patency and are associated with greater otorrhea, suggesting middle ear toxicity.