Twenty 5-fluorouracil-induced temperature-sensitive (ts) mutants of mouse hepatitis virus strain A59 were isolated from 1284 virus clones. Mutants were preselected on the basis of their inability to induce syncytia in infected cells at the restrictive temperature (40 degrees) vs the permissive temperature (31 degrees). Of these mutants, only those with a relative plating efficiency 40 degrees/31 degrees of 3 x 10(-3) or smaller were kept. Virus yields at 40 degrees compared to 37 degrees and 31 degrees (leakiness) were determined. Most mutants (16) were RNA-, i.e., unable to synthesize virus-specific RNA at the restrictive temperature. The other four were RNA+. No qualitative differences were detected in the virus-specific RNAs in cells infected with RNA+ ts-mutants, both at 31 degrees and 40 degrees. Virus-specific proteins present in cells infected with ts-171 (RNA-) and the RNA+-mutants (ts-43, ts-201, ts-209, and ts-279) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates. No qualitative differences in the pattern of virus-specific cellular proteins were detected among the mutants except for an additional polypeptide of about 46,000 daltons in ts-209-infected cells. Finally, the neuropathogenic properties of eight of the mutants were investigated. Whereas 10(2) PFU of wild-type virus injected intracerebrally killed 50 to 100% of 4-week-old Balc/c mice within 1 week, the mutants were highly attenuated. A dose of 10(5) PFU lead to no or transient disease. However, 4 weeks after infection with ts-342, ts-43, or ts-201 obvious histological changes were observed in brain and spinal cord of clinically healthy mice.