One-hundred and seventeen temperature-sensitive (ts) mutants have been isolated from the challenge virus strain (CVS) of rabies virus (RV). The criterion used for this selection was the absence of plaque-forming units on CER cells under agarose medium at the non-permissive temperature (NPT) of 38.5 degrees C. Of these mutants, 102 were induced by 5-fluorouracil. This compound was much more effective as a mutagen than either nitrous acid or the alkylating agents ethyl methanesulphonate and methyl methanesulphonate. Seventy-six of the ts mutants exhibited residual multiplication at NPT which was less than 2% of that of the wild-type virus. Attempts to demonstrate complementation were unsuccessful, although three phenotypic groups have been established by biochemical and immunological tests. Possible reasons for the failure to obtain complementation are discussed.