The availability of high-resolution structural information for HA in its different conformations has made it the best model system for studies on viral as well as cellular membrane fusion proteins. In spite of this, fundamental questions remain unanswered regarding the molecular mechanics of HA-mediated fusion. These include: (1) the molecular determinants responsible for triggering the acid-induced conformational changes required for fusion; (2) the extent to which the HA structural rearrangements must take place for fusion, and the significance of adopting the lowest free energy structure resulting from molecular rearrangement; (3) the length requirements for fusion of the peptide chains that link the rod-like structure of low pH HA to the membrane anchor and fusion peptide domains and; (4) the significance of specific highly conserved fusion peptide residues for the fusion process. We hypothesize that a region proximal to the fusion peptide in the neutral pH HA contains the residues responsible for triggering the acid-induced conformational changes required for fusion, and aim to identify these and assess the contributions of each. We also hypothesize that extrusion of the fusion peptide alone is not sufficient for biologically relevant fusion and that total conversion to the stable form of the molecule represented in the low pH crystal structures is critical for function. We aim to generate mutants to identify residues that are critical both for the structural stability of low pH HA, and for placing the membrane-associating domains in close proximity to one another as a component of the fusion process. We also aim to address distance constraints for fusion between the membrane-associating regions and the end of the low pH rod-like structure. Furthermore, we hypothesize that sequence and structural characteristics along the length of the 23-residue fusion peptide will prove critical for function, and we will continue to focus attention on this region in an effort to understand these considerations. We will address each of these issues using a combination of functional, biochemical, biological, and structural approaches using expressed mutant HAs, fragments of the molecule, and infectious viruses generated by reverse genetics. The elucidation of the questions that we pose will be instrumental in providing a platform for future studies on viral and cellular fusion proteins, and for instigating new ideas regarding antiviral drugs that target membrane fusion function.