Influenza hemagglutinin (HA, strain A/PR/8/34) was purified and reconstituted into supported planar membranes in a two-step process: 1) HA was purified by C12E8 detergent solubilization followed by detergent removal with Biobeads; (2) the purified HA was then incorporated into "viroplanes," i.e. supported planar membranes which contained the viral membrane proteins. This step was accomplished by a spontaneous reaction of the HA-proteoliposomes with a phospholipid monolayer that was supported on a quartz microscope slide. The reconstitution of the HA into the planar membranes was followed by total internal reflection fluorescence microscopy (TIRFM) using fluorescein-labeled HA. By changing the solution concentration of HA, surface concentrations between 2.4 x 10(4) and 4.3 x 10(4) HA monomers/micron 2 were reached. Greater than 90% of all HA molecules were oriented with their ectodomain facing away from the substrate toward the large aqueous compartment of the measuring cell. Binding experiments with conformation-sensitive monoclonal antibodies against HA established that the reconstituted HA could undergo the low pH-induced conformational change in the supported bilayer. Binding of vesicles containing the fluorescent lipid analog N-(7-nitro-2,1,3-benzoxadiazol-4-yl)egg phosphatidylethanolamine was also measured by TIRFM. Vesicle binding was promoted when sialic acid-containing gangliosides or negatively charged lipids were included in these target membranes. Membrane fusion of the HA bound vesicles was monitored by measuring long range (over several micrometers) lateral diffusion coefficients of the lipids in the bound layer by fluorescence recovery after photobleaching. The vesicles did not fuse at pH 7.4, but efficient vesicle fusion occurred on the viroplanes after acidification of the environment with pH 5 buffer. This fusion reaction was only observed when the bound vesicles exceeded a critical threshold surface concentration. The successful reconstitution of membrane fusion sites in a planar supported membrane system opens new possibilities for studying fusion intermediates by localized spectroscopy and microscopy.