A provisional temperature-sensitive mutant, ProTs8, of influenza A/WSN (H0N1) virus was isolated from primary chicken embryo fibroblast culture inoculated with influenza A/WSN (H0N1) and incubated with medium 199, containing 2% chicken embryo extract, and 400 micrograms/ml of 5-fluorouracil. Its EOP (efficiency of plaquing) value between 39.5 degrees C and 33 degrees C was about 0.007, and the control was about 0.82. Through lowering the shut-off-temperature to 38 degrees C, we had successfully isolated two temperature-sensitive mutants, Ts8-38 and Ts8-37, from their parent, ProTs8. 50% mouse virulent doses (MVD50) of these two mutants were around 10(-0.6), while the control group was 10(-4.0). Groups of mice immunized with 0.4 MVD50 or 0.04 MVD50 of Ts8-38 or Ts8-37 mutant by nasal instillation had shown significant resistance to the challenging infection of the wild type virus, influenza A/WSN (H0N1) (challenge dose, 1,000 MVD50 or 100 MVD50), if compared to the control group.