RNA:DNA hybridization was used to study the synthesis of viral RNA in two DNA-minus, temperature-sensitive mutants of type 5 adenovirus (H5ts125 and H5ts149) belonging to two different, non-overlapping complementation groups. Hybridization competition analysis showed that both mutants transcribed all early gene sequences at the restrictive temperature (41 C). In mutant-infected cells at 41 C, the rate of viral transcription was similar to the rate of early RNA synthesis in wild-type virus infection and was dependent on the multiplicity of infection; little or no late transcription was detected. The shutoff of class I early RNA transcription was shown to be a late function during wild-type virus infection and did not occur at 41 C in mutant-infected cells. When mutant-infected cells were incubated at the permissive temperature (32 C) for 25 h and then shifted to 41 C, the rate of viral DNA synthesis decreased rapidly for H5ts125 and slowly for H5ts149. However, the rate of viral transcription remained unchanged in H5ts125-infected cells for at least 3 h after the temperature shift; although the synthesis of viral DNA had stopped by this time, the synthesis of late viral RNA sequences continued.