Ebola virus (EBOV) enters cells from late endosomes/lysosomes under mildly acidic conditions. Entry by fusion with the endosomal membrane requires the fusion loop (FL, residues 507-560) of the EBOV surface glycoprotein to undergo a pH-dependent conformational change. To find the pH trigger for this reaction we mutated multiple conserved histidines and charged and uncharged hydrophilic residues in the FL and measured their activity by liposome fusion and cell entry of virus-like particles. The FL location in the membrane was assessed by NMR using soluble and lipid-bound paramagnetic relaxation agents. While we could not identify a single residue to be alone responsible for pH triggering, we propose that a distributed pH effect over multiple residues induces the conformational change that enhances membrane insertion and triggers the fusion activity of the EBOV FL.