Influenza virus hemagglutinin (HA) and neuraminidase (NA) are known to associate with lipid rafts, membrane microdomains comprised of densely packed cholesterol and sphingolipids. These specialized membrane regions are believed to be involved in the budding of many enveloped viruses including influenza virus. Quantitative analysis of HA distribution on the surface of virus-infected cells by immunogold staining shows an organization into clusters that grow in size as the expression level of HA increases with time post-infection (p.i.) ( approximately 325-500 nm at 4 h p.i. and approximately 425-600 nm at 6 h p.i.). These HA-containing clusters are likely derived from lipid rafts as they contain a high density of the raft marker ganglioside GM1 and are dependent upon the presence of cholesterol. The clustering of HA is an intrinsic property of the HA protein and occurs in the absence of expression of other viral proteins. NA is also found sequestered within the same microdomains as HA, whereas the M2 ion channel protein does not concentrate within the raft-like microdomains. Quantification of the distribution of surface expressed HA by examining serial sections of virus-infected cells suggests that the HA-containing microdomains give rise to regions of influenza assembly and budding.