The ancestral gamete fusion protein, HAP2, catalyzes sperm-egg fusion in a broad range of taxa dating to the last eukaryotic common ancestor. Remarkably, HAP2 orthologs are structurally related to the class II fusogens of modern-day viruses, and recent studies make clear that these proteins utilize similar mechanisms to achieve membrane merger. To identify factors that may regulate HAP2 activity, we screened mutants of the ciliateTetrahymena thermophilafor behaviors that mimicΔhap2knockout phenotypes in this species. Using this approach, we identified two new genes,GFU1andGFU2, whose products are necessary for the formation of membrane pores during fertilization and show that the product of a third gene, namelyZFR1, may be involved in pore maintenance and/or expansion. Finally, we propose a model that explains cooperativity between the fusion machinery on apposed membranes of mating cells and accounts for successful fertilization inT. thermophila’smultiple mating type system.