Cultures of bovine kidney (BK) cells infected with temperature-sensitive (ts) mutants of foot-and-mouth disease virus (FMDV) were incubated at 38.5 degrees C, a temperature nonpermissive for mutant virus growth and RNA synthesis. The cells were subsequently resistant to viral growth and RNA synthesis when superinfected with wild-type FMDV and with heterologous fowl plague virus. The extent of interference was proportional to the multiplicity of infection of the ts mutant. It increased with time elapsed between infection with mutant and challenge infection, becoming greater than 99 percent after 24 hours. Interference was not proportional to decreased levels of cellular protein synthesis. The interference could be produced in the presence of actinomycin D, and thus was apparently mostly caused by the ts mutant itself rather than by interferon. The interference could not be produced in other less susceptible cell lines. Supernatant fluids from the BK cells infected with ts mutant virus interfered with wild-type FMD viral growth and RNA synthesis in fresh BK cells, and also showed low levels of activity in a vesicular stomatitis virus-plaque reduction assay. The properties of the supernatant fluid-interfering agent resembled to some extent those of an interferon. The ts mutant-mediated interference factor was apparently not able to diffuse into the supernatant fluid.