The purpose of this study was to determine whether mitomycin C could be used to prolong the patency of the myringotomy site in the absence of ventilation tubes in rats, We examined the effect of increased exposure time and repeat application of mitomycin C to the myringotomy site, Sixty animals were separated into 4 groups: group A had a single application of mitomycin C for 10 minutes; group B had a single application for 20 minutes; group C received 2 10-minute applications separated by 1 week; and group D received 2 applications (20 and In minutes) separated by 1 week. Mitomycin C and bacteriostatic saline solution were applied to the right and left myringotomy sites in each rat, respectively. Experimental ears in groups A, B, C, and D remained open for a median time of 6.5, 5.5, 6.5, and 8.5 weeks, respectively. The control ears healed within 1.5 weeks. This difference was statistically significant with P < 0.001 for each group. We conclude that mitomycin C is effective in prolonging the patency of myringotomies in rat tympanic membranes. Increased exposure time or repeat application of mitomycin C did not statistically alter the patency rate.