Purpose: The authors compare the success of macular hole surgery with a short duration tamponade using 5% perfluoropropane (C3F8), intermediate duration tamponade using 10% C3F8, or a long duration tamponade using 16% C3F8 in a prospective consecutive clinical trial at two institutions. Methods: Vitrectomy and fluid-gas exchange was performed in 29 eyes treated with 5% C3F8 and 1 week of prone positioning, 30 eyes treated with 10% C3F8 and 2 weeks of prone positioning, and 90 eyes treated with 16% C3F8 and 2 weeks of prone positioning 90% of time, followed by 2 weeks of prone positioning 50% of time. Results: The macular hole was closed successfully in 19 of 29 eyes (65.6%) treated with 5% C3F8, 19 of 30 eyes (63.3%) treated with 10% C3F8, and 85 of 90 eyes (94.4%) treated with 16% C3F8 at 3 months (P = 0.00017 for 5% vs. 16% C3F8, P = 0.00006 for 10% vs. 16% C3F8). The visual acuity improvements measured by refraction using the Early Treatment Diabetic Retinopathy Study (ETDRS) acuity charts paralleled the success of macular hole closure. The acuity improved a mean improvement of + 9.6 ETDRS letters for eyes treated with 5% C3F8, + 6.4 ETDRS letters for eyes treated with 10% C3F8, and + 13.1 ETDRS letters in eyes treated with 16% C3F8. Conclusions: A long duration intraocular gas tamponade with 16% C3F8 gives a substantially higher success rate for macular hole surgery than a short (5% C3F8) or intermediate duration (10% C3F8) intraocular gas tamponade.