Three high temperature-sensitive (ts) mutants of foot-and-mouth disease virus were characterized by their relative abilities to grow at 33 or 38.5 C, to kill infant mice, to infect guinea pigs, and to produce foot-and-mouth disease in steers. Mutants ts-24 and ts-42 did not grow at 38.5 C; both may have produced considerable quantities of noninfectious virus particles at 33 C. A third mutant, ts-22, appeared "leaky" because it multiplied to a limited extent during prolonged incubation at the nonpermissive temperature. Mutant ts-42 was pathogenic for infant mice, whereas ts-22 and ts-24 were essentially apathogenic. Mice were protected against the lethal effects of the wild-type (wt) virus if injected 1 week earlier with ts-22, apparently as a result of specific antibody development. One-half of the guinea pigs injected with the mutant viruses showed lesion development, but only at the site of injection. Antibody development was also much less than in those animals injected with the wt virus. The onset of FMD in steers was delayed and the severity of the disease was diminished after intradermalingual injection of the mutant viruses.