The biochemical properties of the major virion polypeptides (HA1, HA2, M, NP, NA) of 19 influenza A virus strains have been compared by two-dimensional polyacrylamide gel electrophoresis using nonequilibrium pH gradient electrophoresis in the first dimension. The highly variable surface antigen of the virus, the hemagglutinin (HA), exhibited multiple polypeptide subspecies varying extensively in charge. Comparison of the HA of the different influenza A strains demonstrated that most strains exhibit a unique hemagglutinin with distinguishable electrophoresic properties. Differences in charge and/or molecular weight of at least one of the two HA subunits (HA1 or HA2) were found for strains within the same subtype and for serologically indistinguishable strains such as A/USSR/90/77 and A/FW/l/50. Differences in the matrix (M) and neuraminidase (NA) proteins also were observed between strains. The results of this study indicate that the comparative examination of the two-dimensional polypeptide patterns of a particular virus isolate may be useful for the purposes of strain identification, determination of strain purity, homogeneity, and determination of gene origin following experimental or natural recombination events. © 1979.