Sequential immunization with whole inactivated viruses PR8 and FM1 induced minimal original antigenic sin, yet led to diminished protective immunity. BALB/c mice (six mice/group) were i.m. immunized with 1400 HAU of whole formalin-inactivated PR8. Control mice were immunized with PBS. A month later, the PR8-immune and control mice were immunized with 1400 HAU of whole inactivated FM1. Serum samples were collected at memory (day 28) following primary immunization (PR8), and days 7, 14, and 28 following secondary immunization (FM1). Sera were treated with receptor-destroying enzyme II, heat-inactivated, and analyzed for neutralization titers (A) and HAI titers (B) using freshly grown PR8 and FM1 viruses in MDCK cells. A month following FM1 immunization, mice were intranasally challenged with a lethal dose (100 × LD50) of mouse-adapted FM1 virus (C). Mouse lungs were harvested 4 days after challenge and assessed for lung viral titers via plaque assay on MDCK cells; the data are shown as plaque forming units (pfu/ml). Open bars represent serum titers against PR8 and filled bars against FM1. Each data point represents an individual animal. Error bars represent SEM. The data represent three separate experiments. *, p < 0.05; **, p < 0.02.