The reversion of temperature-sensitive (ts) mutants of fowl plague virus to the ts+ phenotype was correlated with pathogenicity for chicken. Two types of ts mutants were investigated: those obtained by mutagenesis with 5-fluorouracil and those obtained by undiluted passages at 33 degrees C. The reversion frequency of the former mutants depended on the RNA segment in which the ts defect was located, mutations in RNA segments 1 and 2 having the highest reversion frequency, those in the RNA segments coding for the glycoproteins the lowest. ts mutants obtained by undiluted passages behaved differently in this respect. There was an approximate correlation between frequency of reversion and pathogenicity for chicken. Double mutants induced by 5-fluorouracil, having one tight and one leaky mutation, reverted easily without loss of the leaky mutation. These double mutants were still to a limited extent pathogenic for the chicken. Only one double mutant with two tight mutations (ts 293) was completely nonpathogenic after intramuscular inoculation. Two ts mutants with multiple tight defects (ts 1/1 and ts 3/18) obtained by undiluted passage did not revert to wild-type after injection into embryonated eggs and incubation at 33 degrees C, but they were still slightly pathogenic for the chicken. There was no obvious correlation between the shut-off temperature and pathogenicity of mutants carrying a single ts defect. However, for mutants with multiple tight mutations a high shut-off temperature seemed to be essential for reversion during serial passages as well as for pathogenicity in the chicken, when different routes of inoculation were examined. ts mutants seem to be safe as live vaccines only, (1) if they carry at least two tight ts defects, (2) if they have a relatively low shut-off temperature, and (3) if they could be administered other than via the respiratory tract.