To investigate the effect of intraoperative mitomycin C (MMC) application on muscle insertion migration after adjustable surgery, hang-back recession was bilaterally performed for 4 mm on the inferior rectus muscle in 30 rabbits. The scleral wound of the insertion site of one eye of each pair was treated with a sponge soaked with a solution cotaining mitomycin at a concentration of either 0.4 or 1.0 mg/ml for 5 min. As a control, the contralateral eye was treated with a distilled-water-soaked sponge. Three, 6 and 12 weeks later, the distance from the anterior border of the reattached muscle to the original insertion was measured with in vivo and microscopic examination. Anterior migration of the muscle insertion was observed in both the MMC-treated and control eye. The measurement by the in vivo and histologic examinations revealed that MMC treatment did not significantly reduce the anterior migration relative to the control eye. Analysis of the time course in the MMC-treated eye revealed a significant increase during the observation period in anterior migration as measured by the histologic examination (p = 0.0267 for the 0.4 mg/ml group and p = 0.0408 for the 1.0 mg/ml group). Exposure to MMC has no significant inhibitory effect on the muscle migration compared to that in the control eyes.