Acid treatment of influenza A and B virus preparations followed by addition of dithiothreitol (DTT) and centrifugation through a sucrose cushion removes the HA1 subunit of hemagglutinin from virus. Rabbit sera made against these subviral particles and untreated virus were tested in a radioimmune precipitation assay using [35S]cysteine-labeled virus. Conditions of the assay permitted discrimination of discrete HA1- and HA2-specific antibody populations. It was found that (a) sera raised to intact influenza A virus preparations contained both HA1- and HA-2 specific antibodies, (b) sera made to subviral particles of influenza A virus contained HA2-specific antibody but had little or no detectable HA1-specific antibody. (c) the HA2-specific antibodies were partially cross-reactive with the HA2 of an influenza A virus of a different subtype, and (d) sera raised against two strains of untreated influenza B viruses contained antibodies which were cross-reactive with the HA2 as well as the NP of influenza A viruses.