Biochemical characterization of 70 temperature-sensitive (ts) mutants of rabies virus has been done by following the appearance of viral proteins and RNA molecules in infected cells at both permissive and nonpermissive temperature. The presence or absence of the nucleocapsid protein (N) was demonstrated by treating infected cells with anti-N fluorescent antibodies. At 33 degrees C, all the mutants induced a fluorescence comparable to the wild type. At 39.6 degrees C, the mutants can be classified into three groups. Three mutants induced a fluorescence comparable to the wild type (F+ mutants); 54 mutants induced a faint fluorescence which was proportional to the multiplicity of infection and increased with time (F+- mutants). No fluorescence could be detected for the 13 remaining mutants (F- mutants). The synthesis of all viral proteins was shown to be normal for F+ mutants, indicating that transcription and replication of the virus were normal and that the ts lesion was located in a protein which is not directly required for those functions. The synthesis of all viral proteins was similarly decreased for F+- mutants and undetectable for the F- mutants. This suggests that the ts lesion affects the transcription and/or replication of the virus. By annealing techniques it was demonstrated that the F+- mutants were able to perform some amount of secondary transcription at nonpermissive temperature. No secondary transcription occurred with F- mutants. When detectable (i.e., at higher multiplicity of infection), primary transcription of F- mutants was normal.