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To compare the efficacy and safety of subtenon injection of mitomycin C (MMC) with that of conventional application of MMC-soaked sponges in trabeculectomy.In this multicentre randomised clinical trial, 80 consecutive open-angle glaucoma cases were randomised into two groups; group 1 received a subtenon injection of 0.1 mL of 0.01% MMC, while group 2 received 0.02% MMC-soaked sponges. Primary outcome measure was intraocular pressure (IOP), and secondary outcome measures were endothelial cell count (ECC) changes and bleb morphology according to the Indiana Bleb Appearance Grading Scale. Outcome measures were compared at 1, 3 and 6 months postoperatively. Complete and qualified success was defined as IOP within 6–15 mm Hg without and with medications at month 6, respectively.Mean preoperative IOP was 21.8±5.1 in group 1, which reduced to 10.3±3.7 mm Hg at final visit (p<0.001). Corresponding values for group 2 were 21.8±5 and 10.8±3.5 mm Hg respectively (p<0.001). Complete success was 82.5% in both groups, and qualified success was 0 and 2.5% in groups 1 and 2, respectively. (p=0.316) The blebs tended to be more diffuse, less vascularised and shallower in group 1, at month 6 (p=0.45,<0.001 and <0.007 respectively). ECCs did not change significantly at final visit (p=0.813).Subtenon injection of MMC is a safe and effective alternative to the conventional soaked sponge method. This method produces more favourable bleb morphology after trabeculectomy.NCT02385370, Post-results.